Peptides for skin rejuvenation and methods of using the same

ABSTRACT

The invention provides compositions for stimulating the formation of one or more extracellular matrix components that contain a lipoaminoacid derivative of the tripeptide carnosine such as N-Octanoyl Carnosine. Also provided are compositions containing N-Octanoyl Carnosine in combination with selected tripeptide and/or tetrapeptides as well as pharmaceutical and/or cosmetic compositions containing such compositions. The invention further provides methods of using the compositions and compositions of the invention to treat, alleviate, and/or ameliorate a symptom, condition, disorder, or disease of the skin or mucosa, wherein the symptom, condition, disorder, or disease is associated with changes in extracellular matrix components.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.14/202,418, filed Mar. 10, 2014, now U.S. Pat. No. 9,375,398, whichclaims priority to U.S. Provisional Patent Application No. 61/779,601,filed Mar. 13, 2013, the disclosure of each of which are hereinincorporated by reference herein in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “27894-507001US_ST25.txt”, which wascreated on Jun. 30, 2014 and is 3 KB in size, is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the fields of skin rejuvenation ofhuman skin, maintenance of healthy skin, restoration of damaged skin,wound healing, treatment of atrophy of any human tissue, and/ortreatment of conditions, disorders, and diseases of the skin and mucosaassociated with changes in extracellular matrix components. Moreparticularly, the invention relates to compositions and methods of usingsuch compositions in order to improve the appearance of aged skin bystimulating extracellular matrix components including, for example,collagens, elastin and hyaluronic acid in humans.

BACKGROUND OF THE INVENTION

Human skin is a complex organ which extends over the entire body. Thereare different types of skin at different portions of the body. Forexample, facial skin is different from that of the scalp, and even theskin on the palm of the hand is different than that on the back of thehand. Although the type of skin can vary over a person's body, skin isgenerally composed of two main layers of tissue. The epidermis, theoutermost layer, is composed of several layers. The dermis, also calledcorium or cutis vera, is composed of a papillary layer above and areticular layer below.

The human epidermis is principally composed of keratinocytes butcontains also other types of cells including the melanocytes and theLangerhans' cells. Each of these cell types contribute, through theirspecific function, to the essential role played by the skin.

The dermis provides a solid support for the epidermis. It is also itsfeeder layer. The dermis consists mainly of fibroblasts but leukocytes,mast cells or tissue macrophages are also present. The dermis furthercontains blood vessels and nerve fibers. The acellular part (i.e., thearea in between the cells) of the dermis is called extracellular matrix.The extracellular matrix of skin is composed of various extracellularcomponents including proteins; in particular collagen fibers andelastin. Other extracellular matrix components of skin includeglycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate, dermatansulfate, keratan sulfate, heparan sulfate, etc.), proteoglycans (e.g.,fibromodulin, decorin, biglycan, perlecan, heparan sulfate proteoglycan2, agrin, versican, aggrecan, lumican, collagen type IX, collagen typeXII, collagen type XIV, testican 1, testican 2, etc.) and variousglycoproteins (e.g., fibrillin 1, thrombospondin-1 and -2, tenascin-Cand —X, osteopontin, fibronectin, laminin-5 and -6, vitronectin, etc.).These extracellular components are synthesized by dermal fibroblasts,which make dermal fibroblasts the primary constituent in the structuralassembly of the dermis.

The extracellular matrix is a highly heterogeneous amalgam ofmorphologically diverse architectural entities. It organizes and impartsstructural integrity to individual tissues, in addition to modulatingcell behavior by interacting with cell surface receptors and solublegrowth factors. Dysfunctions and changes in components of theextracellular matrix can therefore interfere with both tissue integrityand cell performance. Dysfunctions and changes in components of theextracellular matrix of skin and mucosa in humans can lead to skinaging, skin atrophy, damaged skin, wounded skin, atrophy of vulva andvagina (vulvovaginal atrophy), or to any other conditions, disorders anddiseases of skin and mucosa associated with changes in extracellularmatrix components.

Therefore, a need exists in the art for compositions having improvedactivity that maintain or even increase the level of a rather largenumber of extracellular matrix components, including those that arealtered in aged, damaged, wounded, atrophic skin, atrophy of vulva andvagina, or in any other conditions, disorders and diseases of skin andmucosa in humans associated with changes in extracellular matrixcomponents.

SUMMARY OF THE INVENTION

The instant invention provides alternative peptidic compounds (i.e.,peptides such as carnosine peptidic compounds and peptide derivativesand analogues) or appropriate peptidic combinations thereof (i.e.,combinations of peptides, peptide derivatives and analogues), for thecosmetic and pharmaceutical uses.

In particular, provided herein are compositions for stimulating theformation of one or more extracellular matrix components (e.g., collagenI, collagen III, collagen V, collagen VI, collagen VII, collagen XVI,elastin, laminin, hyaluronic acid, fibrillin, heparan sulfateproteoglycan 2, and/or any combination(s) thereof).

For example, such compositions contain a lipoaminoacid derivative of thedipeptide carnosine where the amino-terminus is acylated. This lipoaminoacid derivative may include, but is not limited to, Octanoyl Carnosine(also referred to interchangeably herein as N-Octanoyl Carnosine).

The chemical structure of Octanoyl Carnosine is provided below:

Octanoyl Carnosine differs in structure and biological properties fromcarnosine. The chemical structure of carnosine is provided below:

Octanoyl Carnosine is a lipoaminoacid derivative of carnosine. OctanoylCarnosine is not naturally occurring. Rather, Octanoyl Carnosine is achemically modified carnosine where the amino-terminus (—NH2) of thedipeptide carnosine has been altered with an acylating agent to form anoctanoyl group. This alteration of the amino-terminus providessignificantly different physicochemical properties (e.g., molecularweight, diffusivity, water solubility, lipophilicity, octanol-waterdistribution coefficient, H-bond formation, etc.), chemical properties(e.g., chemical reactivity towards a chemical reactant, chemicalstability, spontaneous hydrolysis, transformation by carnosinase, etc.),metabolic stability (i.e., proteolytic and/or enzymatic degradation),and biological properties (e.g., stimulation of extracellular matrixcomponents including but not limited to collagens I and III; whether ornot related to different enzyme kinetics, different affinities to cellsurface or nuclear receptors, and/or increased diffusivity through cellmembranes or other physiological barriers).

For example, being more lipophilic (i.e., higher octanol-waterdistribution coefficient) than carnosine provides Octanoyl Carnosine ahigher permeability through mammal skin and therefore a higher potencythan carnosine. For example, being more resistant to degradation (i.e.,higher metabolic stability) than carnosine provides Octanoyl Carnosine alonger and more sustained potency than carnosine. For example, being alipoaminoacid derivative of carnosine provides Octanoyl Carnosine thenovel and unexpected biological properties to differently and moreeffectively stimulate the formation of extracellular matrix components;including but not limited to collagens I and III, as compared tocarnosine.

The compositions of the invention may additionally include one or morecarriers, excipients, and/or additional ingredients suitable for topicaladministration and/or subcutaneous administration.

The compositions of the invention are able to improve the general stateof skin and/or mucosa, rejuvenate skin, treat damaged skin or mucosa,improve atrophic tissue including vulvovaginal atrophy, and/or treatother conditions, disorders and diseases of skin and mucosa associatedwith changes in extracellular matrix components in humans.

More specifically, the invention provides peptidic compositions, orappropriate combinations thereof, that are sufficiently effective to beused for the stimulation of the formation of collagen I, collagen III,collagen V, collagen VI, collagen VII, collagen XVI, elastin, laminin,hyaluronan synthase 2, fibrillin 1, heparan sulfate proteoglycan, and/orhyaluronic acid (or combinations thereof). Such compositions can beparticularly used for the treatment of conditions, disorders and/ordiseases of skin and mucosa associated with changes in extracellularmatrix components in humans.

Even more specifically, the compositions of the invention stimulate theformation of one or more of the extracellular matrix componentspredominantly associated with skin aging (e.g., collagen I, collagenIII, collagen V, elastin, hyaluronic acid, and/or any combination(s)thereof). Such compositions can be particularly used for the treatmentof skin aging in humans.

Importantly, the compositions of the invention stimulate the formationof collagen III to a higher degree than the formation of collagen I.Determination of the degree of collagen III and/or collagen I formationis well within the routine level of skill in the art. Because thesecompositions stimulate the formation of collagen III, they can beparticularly used for the treatment of wounded or damaged skin.

Any of the compositions of the invention can additionally contain one ormore additional active ingredients, wherein the combination of allactive ingredients stimulates the formation of hyaluronic acid. By wayof non-limiting example, the one or more additional active ingredientsmay be tripeptides, tetrapeptides, and/or any combinations thereof.Specifically, the compositions may include Octanoyl Carnosine, incombination with the lipoaminoacid derivative of the tripeptide GHK(N-Palmitoyl-GHK), and the tetra-peptide GEKG (SEQ ID NO: 1). Becausethese compositions (or appropriate combinations thereof) stimulate theformation of hyaluronic acid, they can be particularly used for thetreatment of wounded or damaged skin, atrophic skin and mucosa, andvulvovaginal atrophy.

Unappreciated by the art and unexpectedly, it has been found thatcompositions containing the lipoaminoacid derivative of the dipeptidecarnosine, N-Octanoyl Carnosine, stimulate the formation ofextracellular matrix components such as collagen I, collagen III,collagen V, collagen VI, collagen VII, collagen XVI, elastin, laminin,hyaluronic acid, fibrillin, and/or heparan sulfate proteoglycan 2. Infact, compositions containing Octanoyl Carnosine stimulate the formationof the extracellular matrix components which are predominantlyassociated with skin aging, namely collagen I, collagen III, collagenVII, elastin and/or hyaluronic acid.

Furthermore, unappreciated by the art and most unexpectedly,compositions containing Octanoyl Carnosine stimulate the formation ofsignificantly more collagen III than collagen I. Moreover, compositionscontaining Octanoyl Carnosine combined with selected additional tri- andtetra-peptides stimulate formation of hyaluronic acid in a synergisticmanner.

Any of the compositions described herein may be suitable for topicaladministration in humans on skin aged skin, damaged skin, skin aftercosmetic and/or dermatological procedures, atrophic skin, wounded skin,vulva, vagina, atrophic vulva, atrophic vagina, and/or on skin andmucosa associated with changes in one or more extracellular matrixcomponents. Moreover, in some cases, these compositions may be suitablefor subcutaneous administration in humans.

Compositions according to the invention that contain a combination ofOctanoyl Carnosine, selected tripeptides, and selected tetrapeptidesstimulate the formation of extracellular matrix components in skin ormucosa to a greater degree than any one or two of the active ingredientsalone. For example, the combination of these ingredients producessynergistic results.

The invention provides compositions containing at least OctanoylCarnosine. Optionally, the composition may contain Octanoyl Carnosineand at least one additionally substance (e.g., an acceptable carrierand/or excipient) suitable for topical application and/or forsubcutaneous application.

The invention further provides a composition containing at least anN-acyl derivative of carnosine (other than Octanoyl Carnosine), an esterof Octanoyl Carnosine, an ester of carnosine, and/or any combination(s)thereof. Those skilled in the art will recognize that any of thecompositions described herein can include Octanoyl Carnosine, any otherN-acyl derivative of carnosine, an ester of Octanoyl Carnosine, an esterof carnosine, and/or any combination(s) thereof. These compounds arecollectively referred to herein as “derivatives of Octanoyl Carnosine”or “Octanoyl Carnosine derivatives”. The structure of suitable OctanoylCarnosine derivatives that can be used in the compositions of theinvention is provided below:

wherein R1 is a carbon-containing side chain which can be linear orbranched, saturated or unsaturated; and can contain heteroatomsincluding sulfur atoms (S), oxygen atoms (0), nitrogen atoms (N),phosphorus atoms (P), and/or halogen atoms (F, Cl, Br, I); and

wherein R2 is either a proton atom (H), or a carbon-containing sidechain which can be linear or branched, saturated or unsaturated; and cancontain heteroatoms including sulfur atoms (S), oxygen atoms (O),nitrogen atoms (N), phosphorus atoms (P), and/or halogen atoms (F, Cl,Br, I).

Preferably, the R1 group is a linear, saturated carbon-containing sidechain of —(CH2)n-CH3 whereas n is an integer number between 0 to 18.Preferably, the R2 group is either H, or a linear, saturatedcarbon-containing side chain of —(CH2)m-CH3 whereas m is an integernumber between 0 to 19.

For example for Octanoyl Carnosine, R1=—(CH2)6-CH3 and R2=H. OctanoylCarnosine is the most preferred derivative of carnosine for use in thecompositions and methods of the invention.

Also provided are compositions containing at least Octanoyl Carnosine;in combination with the lipoaminoacid derivative of the tri-peptide GHK,N-Palmitoyl-GHK (also referred to interchangeably herein asPalmitoyl-GHK); and the tetra-peptide GEKG (SEQ ID NO: 1). Morespecifically, also provided are compositions containing OctanoylCarnosine, in combination with Palmitoyl-GHK, GEKG (SEQ ID NO:1), and atleast one additional substance (e.g., carrier and/or excipient) suitablefor topical application.

The invention also provides compositions containing at least OctanoylCarnosine, an N-acyl derivative of carnosine other than OctanoylCarnosine, an ester of Octanoyl Carnosine, and/or an ester of carnosine;in combination with Palmitoyl-GHK, an N-acyl derivative of GHK otherthan Palmitoyl-GHK, and/or an ester derivative of Palmitoyl-GHK; andGEKG (SEQ ID NO: 1), an N-acyl derivative of GEKG (SEQ ID NO: 1), anester derivative of GEKG (SEQ ID NO: 1), and/or an N-acyl GEKG (SEQ IDNO: 1) ester.

Those skilled in the art will recognize that any of the compositionsdescribed herein can include Palmitoyl-GHK, an N-acyl derivative of GHKother than Palmitoyl-GHK, an ester derivative of Palmitoyl-GHK, and/orany combination(s) thereof. These compounds are collectively (andinterchangeably) referred to herein as “derivatives of Palmitoyl-GHK” or“Palmitoyl-GHK derivatives.

Likewise, those skilled in the art will also recognize that any of thecompositions described herein can include GEKG (SEQ ID NO: 1), an N-acylderivative of GEKG (SEQ ID NO: 1), an ester derivative of GEKG (SEQ IDNO: 1), an ester derivative of N-acyl GEKG (SEQ ID NO: 1), and/or anycombination(s) thereof. These compounds are collectively (andinterchangeably) referred to herein as “derivatives of GEKG (SEQ ID NO:1)” or “GEKG (SEQ ID NO: 1) derivatives”.

In addition, those skilled in the art will also recognize that any ofthe compositions described herein can also include cyclic di-, tri,and/or tetrapeptides according to the invention. Cyclic peptides can beobtained by either linking side chains of the peptide or ends of thepeptide sequence (head-to tail, N-backbone to N-backbone, end toN-backbone, end to side chain, side chain to N-backbone, side chain toside chain) through disulfide (disulfide-bond cyclization), lanthionine,dicarba, hydrazine or lactam bridges.

Any of the compositions described herein can also include di-, tri,and/or tetrapeptides according to the invention where the natural aminoacid residue was substituted by unnatural amino acid (D-configuration),an N-methyl amino-alpha-amino acid, a non-proteogenic constrained aminoacid, or a beta-amino acid.

Those skilled in the art will also recognize that any of thecompositions described herein can also include di-, tri, and/ortetrapeptides according to the invention where the amid bond between twoamino acids is replaced by NH-amide alkylation; or as further describedin Drug Discovery Today 2010, 15, 40-56, or in other references known inthe art.

Thus, the present invention relates to compositions containing OctanoylCarnosine (or one or more derivatives thereof), formulated incombination with one or more additional substances, which can include,for example, an acceptable carrier and/or excipient suitable for topicalapplication and/or for subcutaneous application. Optionally, any of thecompositions described herein may also include one or more additionalsubstances with biological activities.

In one aspect, the present invention provides compositions containingOctanoyl Carnosine (or one or more derivatives thereof) in combinationwith Palmitoyl-GHK (or one or more derivatives thereof) and GEKG (SEQ IDNO: 1) (or one or more derivatives thereof) at a weight ratio of 4:1:5,formulated in combination with one or more additional substances whichcan include for example, a carrier and/or excipient suitable for topicalapplication and/or subcutaneous administration. Optionally, suchcompositions may also include one or more additional substances withbiological activities.

Preferably, the compositions of the invention contain at least OctanoylCarnosine, Palmitoyl-GHK, and GEKG (SEQ ID NO: 1) at a weight ratio of4:1:5. However, those skilled in the art will recognize that otherratios of active ingredients can also be used and that determination ofan appropriate weight ratio can be routinely determined.

Moreover, any of the compositions described herein can include betweenabout 0.0001% to 10% per weight of Octanoyl Carnosine, depending on thesolubility of Octanoyl Carnosine in the composition. Such compositionsmay also contain Octanoyl Carnosine, Palmitoyl-GHK, and GEKG (SEQ IDNO: 1) (or one or more derivatives thereof) at a weight ratio of about4:1:5, depending on the solubilities of Octanoyl Carnosine,Palmitoyl-GHK, and GEKG (SEQ ID NO: 1) (or one or more derivativesthereof) in the composition.

For example, some compositions according to the invention contain about0.004% per weight of Octanoyl Carnosine; about 0.001% per weight ofPalmitoyl-GHK, and about 0.005% per weight of GEKG (SEQ ID NO: 1) alongwith one or more acceptable carriers or excipients suitable for topicaladministration in humans on skin, aged skin, damaged skin, skin aftercosmetic and/or dermatological procedures, wounded skin, atrophic skin,atrophic mucosa, atrophic vulva, atrophic vagina, and/or for skin andmucosa associated with changes in extracellular matrix components.

The combination compositions will stimulate production of a biomoleculewhereas application of either of Octanoyl Carnosine, the tripeptidePalmitoyl-GHK, or the tetrapeptide GEKG (SEQ ID NO: 1) (or anyderivatives thereof) alone would not or would stimulate production to asignificantly lesser extent (e.g., amount) than the combination.Alternatively (or additionally), the combination compositions stimulateproduction of a greater extent of a biomolecule than that achieved fromthe peptides when added or administered individually. Indeed, in certainpreferred aspects of the present invention, the combination of OctanoylCarnosine, the tripeptide, and the tetrapeptide (or any derivativesthereof) produces synergistic results.

Also provided herein are pharmaceutical and/or cosmetic compositionsand/or formulations containing any of the compositions of the inventionalong with one or more pharmaceutically and/or cosmetically acceptablecarriers. In these pharmaceutical and/or cosmetic compositions and/orformulations, the composition may contain the combination of OctanoylCarnosine, the lipoaminoacid derivative of the tripeptide GHK (i.e.,N-Palmitoyl-GHK) and the tetra-peptide GEKG (SEQ ID NO: 1). By way ofnon-limiting example, these components of the composition and/orformulation may be present at a weight ratio of 4:1:5.

The invention further provides kits containing, in one or morecontainers, any of the pharmaceutical and/or cosmetic compositionsand/or formulations of the invention.

The compositions of the present invention are particularly suitable forskin rejuvenation or improving the appearance of aged skin. In addition,these compositions can also be used for maintaining healthy skin,restoring damaged skin, enhancing the restoration of skin after cosmeticand dermatological procedures, wound healing including scarless woundhealing, treating atrophy of any human tissue including vulvovaginalatrophy, and for other conditions, disorders and diseases of skin andmucosa in humans associated with changes in extracellular matrixcomponents.

These compositions for skin rejuvenation can be used for improving theappearance of aged skin, maintaining healthy skin, restoration ofdamaged skin, enhancing the restoration of skin after cosmetic anddermatological procedures, wound healing including scarless woundhealing, treatment of atrophy of any human tissue including vulvovaginalatrophy, and for other conditions, and/or disorders and diseases of skinand mucosa in humans associated with changes in extracellular matrixcomponents.

Certain aspects of the present invention also relate to the use of suchcompositions to make cosmetics, personal care products, feminine careproducts, hygiene products, dermatology products, pharmaceuticalpreparations, or medicaments for maintaining healthy skin, skinrejuvenation, restoration of damaged skin including, but not limited to,skin after cosmetic and dermatological procedures, wound healing,treatment of atrophy of any human tissue including vulvovaginal atrophy,and/or other conditions, disorders and diseases of skin and mucosa inhumans associated with changes in extracellular matrix components.

This is accomplished by topical application of the composition of theinvention to the skin or mucosa of the human needing such treatment. Insome limited cases, this can be accomplished by subcutaneousadministration of the composition of the invention in a human needingsuch treatment.

Certain aspects of the present invention also relate to methods of usingsuch compositions to improve the state and appearance of human skin andto prevent and/or reduce the visible signs of aging and/or for enhancingthe restoration of skin after cosmetic and dermatological procedures, aswell as for enhancing wound healing, reducing the atrophy of any humantissue including vulvovaginal atrophy, and for improving otherconditions, disorders and diseases of skin and mucosa in humansassociated with changes in extracellular matrix components. Thesemethods generally involve topically applying the composition to theaffected skin or the affected mucosa when needed, in the amount and atthe frequency best suited for the purpose. Methods of preventing,delaying the onset, or treating a skin or mucosal condition, disorder ordisease are also contemplated.

Furthermore the present invention relates also to the use of OctanoylCarnosine (or any derivatives thereof), or any peptide combinations withOctanoyl Carnosine (or any derivatives thereof), as active agents inmedicine (i.e., active pharmaceutical ingredients) to make a medicament.

Among the additional advantages of using peptide Octanoyl Carnosine (orany derivatives thereof) and peptide combinations thereof, are the factthat the peptides are less toxic in comparison to the commonly useddrugs for certain indications mentioned herein and that the peptideshave fewer side effects, can be used for a long term treatment ofconditions, disorders and diseases of skin and mucosa in humansassociated with changes in extracellular matrix components, and can beeasily administered as composition suitable for topical application.Moreover, no toxic or noxious degradation products of peptides areformed.

Furthermore, di-, tri-, and tetra-peptides are small (e g, contain onlytwo to four amino acids) and have therefore the advantage of not beingor being less immunogenic; in contrast to larger peptides (i.e.,peptides containing of five or more amino acids) which can becomeimmunogenic and cause allergic reactions. This makes the di-peptideOctanoyl Carnosine, the tri-peptide Palmitoyl-GHK and/or thetetra-peptide GEKG (SEQ ID NO: 1) (or any di-, tri-, and tetra-peptidederivatives thereof) particularly suitable for human use.

Also, di-, tri-, and tetra-peptides are of a lower molecular weight(grams per mol) than the peptides consisting of five or more aminoacids, which provides di-, tri-, and tetra-peptides with a smallermolecular volume than larger peptides, which enables di-, tri-, andtetra-peptides to better (i.e., faster, to a larger extent) absorb andpenetrate skin, mucous membranes, cell membranes, and/or otherphysiological barriers. Thus, the di-peptide Octanoyl Carnosine, thetri-peptide Palmitoyl-GHK and/or the tetra-peptide GEKG (SEQ ID NO: 1)(or any di-, tri-, and/or tetra-peptide derivatives thereof) areparticularly suitable for topical application.

In particular, repeated topical application of Octanoyl Carnosine (orany derivatives thereof), or preferred combinations thereof withPalmitoyl-GHK and GEKG (SEQ ID NO: 1) (or any derivatives thereof) inaccordance to the present invention can offer the advantages andqualities described herein, as well as others which can be routinelydetermined. By way of non-limiting example, these advantages may includethe ability (in some cases and with certain preferred combinations) toimprove the visible signs of aging in human skin (including, forexample, fine lines, wrinkles, skin folds, enlarged pores, roughness,dryness, loss of elasticity, loss of volume), improve other skin texturedefects such as stretch marks (as caused by pregnancy, trauma or otherinfluences), reduce bags under the eyes (also called “puffy eyes”),reduce dark (under eye) circles (both caused by thinning of the skin,insufficient blood circulation and slack tissue), and as well to reducethe severity of atrophic skin and mucosa including vulvovaginal atrophy.Moreover, some of these compositions have been discovered to have abenefit in wound healing and tissue regeneration.

Provided herein are methods of treating, alleviating, or ameliorating asymptom, condition, disorder, or disease of the skin or mucosa, whereinthe symptom, condition, disorder, or disease is associated with changesin extracellular matrix components, the method comprising administeringan effective amount of any of the compositions and/or formulations ofthe invention to a person (e.g., patient) in need thereof. For example,wherein the symptom, condition, disorder, or disease of the skin ormucosa associated with changes in extracellular matrix components isselected from the group consisting of wounds, aging, age-associateddisorders and diseases, atrophy of any human tissue, disorders anddiseases of vulvar tissue, disorders and diseases of vaginal tissue,and/or any combination(s) thereof.

In some examples, treating, alleviating, or ameliorating the woundresults in scarless wound healing. In other examples, treating,alleviating, or ameliorating atrophy of any human tissue involves thetreatment of vulvovaginal atrophy. In other examples, treating,alleviating, or ameliorating skin and mucosa associated with changes inextracellular matrix components comprises the treatment of vulvodynia,lichen sclerosus, vulvar lichens planus, erosive lichen planus, vulvareczema, vulvar lichen simplex chronicus, ulcers of the vulva, Behcet'sdisease, vulvar intraepithelial neoplasia, and/or any combination(s)thereof.

Any of the methods described herein may involve repeated topicaladministration of any of the compositions and/or formulations describedherein to the patient. By way of non-limiting example, thecompositions/and or formulations may be administered to the patient atleast once or twice a day for at least 30 days.

Alternatively, any of the methods described herein may involve repeatedsubcutaneous administration of any of the compositions and/orformulations described herein to the patient. By way of non-limitingexample, the compositions/and or formulations may be administered to thepatient at least once or twice a day for at least 30 days.

Those skilled in the art will recognize that the administration of anyof the compositions and/or formulations of the invention treats,alleviates, or ameliorates one or more visible signs of aging associatedwith changes in extracellular matrix components selected from the groupconsisting of fine lines, wrinkles, enlarged pores, roughness, dryness,loss of elasticity, loss of volume, atrophic skin, atrophic vulva,atrophic vagina, stretch marks, puffy eyes, dark (under eye) circlescaused by thinning of the skin, dark (under eye) circles caused byinsufficient blood circulation and slack tissue, and any combinationsthereof.

Those skilled in the art will further recognize that the administrationof any of the compositions and/or formulations of the invention treats,alleviates, or ameliorates conditions, disorders and diseases of skinand mucosa in humans associated with changes in extracellular matrixcomponents which are not related to aging and which are selected fromthe group of wounded skin, damaged skin after cosmetic anddermatological procedures, atrophy of skin and mucosa due to othercauses than aging (e.g., emotional stress, use of oral contraceptivepills, use of aromatase inhibitors, due to surgery, etc.), and for otherconditions, and/or disorders and diseases of skin and mucosa in humansassociated with changes in extracellular matrix components which are notrelated to aging.

Also provided are methods of improving or rejuvenating the appearance ofskin comprising administering an effective amount of any of thecompositions and/or formulations of the invention to a patient in needthereof. By way of non-limiting example, the compositions and/orformulations are administered to the patient in the form of a cosmetic,a personal care product, a feminine care product, a hygiene product, adermatology product, a pharmaceutical preparation, a medicament, or anycombination thereof. Such compositions and/or formulations can beadministered topically to the skin or mucosa of the patient and/orsubcutaneously to the patient.

The invention also provides methods of maintaining healthy skin or ofpreventing, ameliorating, or delaying aging of skin, the method byadministering an effect amount of any of the compositions and/orformulations described herein to a patient in need thereof. For example,the compositions and/or formulations may be administered to the patient(i.e., topically to the skin or mucosa) in the form of a cosmetic, apersonal care product, a feminine care product, a hygiene product, adermatology product, a pharmaceutical preparation, a medicament, or anycombination thereof. In such methods, administration of the compositionsand/or formulations prevents, ameliorates, or delays the development ofone or more signs of aging selected from the group consisting of finelines, wrinkles, enlarged pores, roughness, dryness, loss of elasticity,loss of volume, atrophic skin, atrophic vulva, atrophic vagina, stretchmarks, puffy eyes, dark (under eye) circles caused by thinning of theskin, dark (under eye) circles caused by insufficient blood circulationand slack tissue.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing one way of the synthesis and purificationof Octanoyl Carnosine (Octanoyl-beta-Ala-His-OH) in its acetate saltform by solid-phase synthesis.

FIG. 2 is a schematic showing one way of the synthesis and purificationof Palmitoyl-GHK (Palmitoyl-Gly-His-Lys-OH) in its acetate salt form bysolid-phase synthesis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be better understood from the followingdescription.

In this specification where reference is made to particular features ofthe invention it is to be understood that the disclosure of theinvention in this specification includes all appropriate combinations ofsuch particular features. The embodiments disclosed in thisspecification are exemplary and do not limit the invention. As used inthis specification, the singular forms “a”, “an”, and “the” includeplural reference unless the context clearly dictates otherwise. Theterms “comprises” and “contains” and grammatical equivalents thereof areused in this specification to mean that, in addition to the featuresspecifically identified, other features are optionally present. The term“at least” followed by a number is used herein to denote the start of arange beginning with that number.

This specification incorporates by reference all documents referred toherein and all documents filed concurrently with this specification orfiled previously in connection with this application, including but notlimited to such documents which are open to public inspection with thisspecification. All publications cited herein are hereby incorporated byreference in their entirety.

While the specification concludes with the claims particularly pointingand distinctly claiming the invention, it is believed that the presentinvention will be better understood from the following description. Theterms “having” and “including” are to be construed as open-ended unlessthe context suggests otherwise.

Extracellular Matrix Components

Collagen occurs in many places throughout the body. So far, at least 28types of collagen have been identified and described which provide avariety of structural and functional properties that collagen exhibitsthroughout the body. The five most common types are collagen I, II, III,IV and V. However, over 90% of the collagen in the body is of type I.

In human skin, collagen types I and III are the predominant types ofcollagen. They are present as fibrils and are responsible for thesolidity and the strength of the dermis. Since type I collagen is thepredominant collagen in adult human skin, comprising about 80% of thetotal bulk of collagen, it plays a major role in providing tensilestrength to the skin. It is clear, however, that type III collagen,which comprises some 10% of the total dermal collagen, also plays acritical role in providing additional tensile properties to the skin andother tissues. (See Journal of Dermatological Science, 24, Suppl. 1,2000, S60-S69).

Structurally, three collagen polypeptides wrap around each other in ahelix to form a triple helix collagen I or III molecule. These moleculesare packed in a five-stranded rope-like structure wherein each collagenmolecule is quarter-staggered with respect to the next to form amicrofibril. Microfibrils are subsequently wrapped around othermicrofibrils to form fibrils, which in turn wrap around other fibrils toproduce even larger fibers.

Using histological and ultrastructural approaches in the past, it hasbeen well-described that chronologically aged skin manifests in areduced synthesis of both collagen types I and III. With respect tophotoaging, Schwarz et al. (Photochem Photobiol 1993, 58, 841-844)demonstrated that the loss in collagen in sun-damaged human skin is dueto increased degradation of both types I and III collagens.Additionally, it was shown that fibroblasts derived from sun-exposedskin synthesize a lower proportion of collagen III than cells fromsun-protected skin. (See J Photochem Photobiol B. 1995, 27: 33-38).

Other collagen types are also present in skin, and a few of them havebeen described to change with skin aging. For instance, collagen VII,which is responsible for anchoring the basement membrane onto dermalmatrix, decreases with aging. (See Eur J Dermatol 2008; 18: 297-302).One of the additional major morphological features of aged skin is analtered dermal epidermal junction which structurally manifests asflattening of the dermal epidermal junction outline with the loss ofrete pegs and re-duplication of the lamina densa. Since the dermalepidermal junction is involved in the cohesion between the dermis andepidermis, age-related alterations in the dermal epidermal junction as aresult of collagen VII decrease entail functional changes in skinresistance to mechanical stress and tissue homeostasis. This maycontribute to wrinkle formation.

Collagen V assembles into diverse molecular forms and studies indicatedthat it is expressed in skin as different subtypes with important butdistinct roles in matrix organization and stability. (See J InvestDermatol 2012, 132: 1841-1849). Whereas collagen V is the defectiveproduct in most cases of classical Ehlers-Danlos syndrome, which is anextracellular matrix component disorder typically characterized by skinfragility and abnormal wound healing, it does not seem to significantlychange with skin aging.

Further collagen types such as collagens VI, XIV and XVI are alsoexpressed in the collagen-rich dermis. Although the structural featuresof these collagens are now well-characterized, their functions stillremain mostly elusive.

The production of collagen in vivo requires activation of the collagenbiosynthesis pathway by which transcription in the cell nucleus promotespolypeptide synthesis via translation from mRNA, organization of thepolypeptides into a pro-collagen triple helix in the cytoplasm,secretion of pro-collagen from the cell, and then cleavage reactions,fibril assembly, and cross-linking extracellularly. Unlike many proteinsthat are stored in secretory granules and then secreted from the cellupon demand, collagen is secreted continuously.

Alterations in content and structure of collagen and other components ofthe extracellular matrix, including but not limited to, elastin andhyaluronic acid are characteristic of aged human skin.

Elastin is a vital component of the extracellular matrix of vertebrates,and provides exceptional properties including elasticity and tensilestrength to many tissues and organs including the skin. Mature elastinis an insoluble and extremely durable protein that undergoes littleturnover, but sustained exposure to proteases may lead to irreversibleand severe damage, and thus to functional loss of the elastic fibernetwork. In general, elastin content decreased with age in sun unexposedskin (i.e., buttock) (i.e., elastin content decreased by about 44%between 50 and 70 years of age). A similar decrease was seen in severesun-exposed skin (i.e., face) (i.e., elastin content decreased by about31% between 50 and 70 years of age). Interestingly, the elastin contentin moderately sun-exposed areas (i.e., forearm) did not significantlychange during aging. This phenomenon might be explained by a combinationof age-induced reduction and sun-dependent increase in elastin, whatappears to be at least partially regulated by UV-induced deposition oflysozyme in elastin fibers. (See JEADV 2006, 20, 980-987).

Fibrillins (e.g., fibrillin 1) are ubiquitous glycoproteins of theextracellular matrix that self-polymerize into filamentous microfibrilsin which individual molecules are organized in longitudinal head-to-tailarrays and associate laterally as well. (See Fibrogenesis & TissueRepair 2010, 3, 24). Fibrillin microfibrils can additionally serve asthe structural template for tropoelastin deposition and/or crosslinkingduring elastic fiber formation. Specific segments of the fibrillinsinteract in vitro with numerous extracellular signaling and cell surfacemolecules, including fibronectin, fibulins, syndecans and integrins. Themultiple molecular interactions of fibrillins are believed to drive theassembly of morphologically distinct macroaggregates, which contributeto imparting the structural integrity to individual tissues and organs(structural role), and to target TGF-beta and BMP complexes to thearchitectural matrix, which contributes to instructing the behavior ofcells (instructive role). TGF-betas and BMPs are potent modulators ofextracellular matrix metabolism that are under the control of a complexnetwork of relays and servomechanisms operating within and outside thecell, and at the cell surface. Thus, fibrillins are important componentsof the extracellular matrix which are necessary for the formation ofother extracellular matrix components such as elastin and elastic fiberformation.

Hyaluronic acid (also called hyaluronan) is an anionic, non-sulfatedglycosaminoglycan. It is unique among glycosaminoglycans since it isnon-sulfated and can be very large, with its molecular weight (g mol-1)often reaching the millions. As a predominant voluminous molecule,hyaluronic acid is a major component of the extracellular matrix of theskin. It provides structure, volume (which is associated with hyaluronicacid's excellent water holding properties), and organization (e.g.,facilitates the transport of ion solutes and nutrients) but alsocontributes significantly to cell proliferation and migration in thedermis. In addition, through the water-attaining properties ofhyaluronic acid, it contributes to the hydration of the skin.

Glycosaminoglycans (e.g., hyaluronic acid, chondroitin sulfate, heparansulfate, dermatan sulfate, keratan sulfate, etc.) and particularlyhyaluronic acid are major components of the cutaneous extracellularmatrix involved in wound healing and tissue regeneration. Wound healingis a dynamic interactive process involving many precisely interrelatedphases, overlapping in time and leading to the restoration of tissueintegrity. The healing process reflects the complex and coordinated bodyresponse to tissue injury resulting from the interaction of differentcell types and extracellular matrix components. Hyaluronan plays a keyrole in each phase of wound healing by stimulating cell migration,differentiation, and proliferation as well as regulating extracellularmatrix organization and metabolism. Glycosaminoglycans and particularlyhyaluronic acid are also involved in skin aging.

As summarized by R. Stern in 2010 (Textbook of Aging Skin, Springer(incorporated herein by reference)), though dermal hyaluronic acid isresponsible for most hyaluronic acid in skin, epidermal cells (e.g.;keratinocytes) are also able to synthesize hyaluronic acid. The mostdramatic histochemical change observed in aged skin is the markeddecrease in epidermal hyaluronic acid A. In skin of an elderly person,hyaluronic acid is still present in the dermis, while the hyaluronicacid of the epidermis has disappeared entirely. The reasons for thisprecipitous fall with aging are unknown. It has been described that thesynthesis of epidermal hyaluronic acid is influenced by the underlyingdermis, thereby indicating that epidermal hyaluronic acid is underseparate controls from dermal hyaluronic acid.

In contrast with previous in vitro and in vivo observations, studiesdocument that the total level of hyaluronic acid remains about constantin human skin with aging. However, the major age-related change is theincreasing avidity of hyaluronic acid with tissue structures with theconcomitant loss of hyaluronic acid extractability. Such intercalatedhyaluronic acid may result in a diminished ability to take on water,what results in a decreased volume and, obviously, a loss in skinmoisture. Progressive loss in the size of the hyaluronic acid polymer inskin as a function of age has also been reported. The increased bindingof hyaluronic acid with tissue as a function of age parallels theprogressive cross-linking of collagen and the steady loss of collagenextractability with age. Each of these phenomena contributes to theapparent dehydration, atrophy, and loss of volume and elasticity thatcharacterizes aged skin.

Other than in skin aging, collagens I and III are also majorextracellular matrix components involved in scar formation. Scarringoccurs after trauma, injury or surgery to any tissue or organ in thebody. Such scars are a consequence of a repair mechanism that replacesthe missing normal tissue with an extracellular matrix consistingpredominantly of collagen types I and III as well as fibronectin andsome other extracellular matrix components. Scarring representsimperfect tissue regeneration. Whereas skin wounds on early mammalianembryos (e.g., up to about 24 weeks of gestation in humans) healperfectly with no signs of scarring and complete restitution of thenormal skin architecture, postnatal wounds heal with scars. (See Dang Cet al., Clin Plast Surg 2003: 30, 13-23).

There are phenotypic differences between the collagen content andcross-linking patterns in fetal and postnatal wounds (See Clin PlastSurg 2003, 30, 13-23 and Curr Opin Pediatr 2012, 24, 371-378). In fetalwounds, type III collagen is rapidly deposited in a fine reticularnetwork that is indistinguishable from uninjured skin. Post-natally, theratio of type I to type III collagen in wounds increases. Of the manydifferent types of collagen identified, fetal skin is known to contain agreater proportion of type III collagen, whereas adult skin consistspredominantly of type I collagen. The predominance of type I collagen inpostnatal wounds provides regenerating tissue with more strength andrigidity. Early scar formation in late gestation fetal woundsdemonstrates larger collagen fibers with greater interfiber space.

In addition, fetal skin generally contains more hyaluronic acid thanadult skin. Further, the hyaluronic acid content of the extracellularmatrix in scarless fetal wounds is increased more rapidly than in adultwounds. Because fetal skin contains more hyaluronic acid than adultskin, several investigators have therefore proposed also a role ofhyaluronic acid in scarless healing. The precise mechanisms of scarlesshealing remain unknown, however, despite the great increase in knowledgegained over the past decade.

The importance of extracellular matrix components collagen I, collagenIII, collagen V, elastin and hyaluronic acid in the skin and theimportance of maintaining, or even increasing, the amount thereof, thus,are self-evident for skin rejuvenation and maintaining healthy skin. Inaddition, collagen I, collagen III and hyaluronic acid have beenparticularly recognized to be important in wound healing and restorationof damaged skin without scar formation.

Conditions Associated with Changes in Extracellular Matrix Components

All terms such as “skin aging”, “signs of skin aging”, “topicalapplication”, and the like are used in the sense in which they aregenerally and widely used in the art of developing, testing andmarketing cosmetic and personal care products, as well as formedicaments which are indicated for skin aging (e.g., cream withtretinoin).

Skin aging is classified into intrinsic aging and extrinsic agingdepending on its cause. Intrinsic aging is a process by which the skinstructure and the physiological functions of the skin deteriorateregardless of environmental changes as a human gets older. Extrinsicaging is caused by continuous exposure to external environment such assunlight and air pollutants. Especially, skin aging caused by sun lightis called photoaging. Ultraviolet (UV) light from the sun is the maincause of the physiological and morphological changes in aged skin.

As intrinsic skin aging proceeds, the skin becomes dry, and fine linesand wrinkles form which become more visible and deepen with age.Further, because of structural and functional changes of the epidermisand the dermis, the skin loses its elasticity and looks drooping. Thedermis becomes thinner and well visible skin folds (e.g., nasolabialfold) form with age. It is estimated that the total quantity of collagenlost each year for adults is about 1%. In addition, the remainingcollagen fibers gradually become thicker, while the cross-linking of thecollagen fibers increases, so that the solubility, elasticity and likethereof decrease. Furthermore, elastin fibers become thicker and thecross-linking thereof also increases. Moreover, the proliferativeactivity of fibroblasts in the dermis decreases with time, and theability of the aging fibroblasts to form (i.e., synthesize) newcollagen, elastin, hyaluronic acid and other components of theextracellular matrix also decreases.

Continuous exposure to the sun is the main cause of extrinsic aging ofskin. The UV component of sunlight, particularly UVA and UVB, isgenerally believed to be the principal causative agent in this processcalled photoaging. The extent of UV exposure required to cause“photoaging” is not currently known, although the amount sufficient tocause erythema (reddening, commonly described as sunburn) in human skinis quantified as the “minimal erythema dose” (MED) from a given UV lightsource. Repeated exposure to sunlight UV at levels that cause erythemaand tanning are, nevertheless, commonly associated with photoaging.

There is a difference between the physiology of intrinsically-aged(i.e., chronologically-aged) skin in comparison with that of photoagedskin. Chronologically-aged skin typically maintains a smooth andunblemished appearance, in comparison with the leathery, blotchy, andoften deep wrinkling of photoaged skin. Photoaging is characterizedclinically by coarseness, wrinkles, mottled pigmentation, sallowness,laxity, telangiectasia, lentigines, purpura and relative ease ofbruising, atrophy, depigmented areas, eventually premalignant, andultimately malignant neoplasm (i.e., abnormal mass of tissue as a resultof neoplasia, which is the abnormal proliferation of cells). Photoagingcommonly occurs in skin that is generally exposed to sunlight such asthe face, ears, bald areas of the scalp, neck, décolleté, forearms, andhands.

Although the typical appearance of photoaged and chronologically agedhuman skin can be readily distinguished, recent evidence indicates thatchronologically aged and UV-irradiated skin share important molecularfeatures including altered signal transduction pathways that promotematrix-metalloproteinase expression (e.g., collagenase, gelatinase)causing extracellular matrix degradation, decreased collagen formation,and alteration or damage to extracellular matrix of skin such as theaccumulation of amorphous elastin-containing material residing beneaththe epidermal dermal junction. This concordance of molecular mechanismssuggests that UV irradiation accelerates many key aspects of thechronological aging process in human skin.

Moreover, in women after menopause, the collagen amount and the skinthickness gradually decrease what causes a sensation of dry or tightskin and a marked increase in skin fine lines and wrinkles. In fact, inaddition of wrinkling and loss of elasticity, aging of the skin is alsoassociated with skin thinning (called atrophy) and delayed woundhealing. These undesirable aging effects are exacerbated by decliningestrogen levels in postmenopausal women. (See Am J Clin Dermatol 2011,12, 297-311). Estrogens (e.g., 17-beta-estradiol) stimulate keratinocyteproliferation, leading to a thicker epidermis, and the formation ofcollagens and other extracellular matrix components. Estrogen productscan be therefore used for the prevention and treatment of skin aging dueto estrogen decline with menopause. Although topical estrogen productsand systemic estrogen replacement therapy has been shown to improve someaspects of postmenopausal skin, long-term estrogen treatment has beenassociated with significant harmful systemic effects such as increase ofbreast cancer rate and cardio-toxic events). Therefore, a need for safeand effective, non-hormonal alternatives for the treatment ofpost-menopausal skin exists.

Furthermore, vulvar-vaginal atrophy (also called vulvovaginal atrophy)is another common consequence of menopause women. (See Adv Nurse Pract2010, 18, 31-32, 34, 55). Vulvo-vaginal atrophy often manifests withdiscomfort what can be experienced as dryness, lack of lubrication,rawness, burning, irritation, inflammation, atrophic vaginitis, andpain. This can ultimately lead to sexual dysfunction. Today, hormonetherapy is the only treatment approved by the US Food and DrugAdministration for vulvovaginal atrophy. Because both physicians andwomen are concerned with the tolerability and safety profile of hormonal(i.e., estrogen, estrogen plus progestin) treatments, alternative,non-hormonal menopause therapies for the treatment of vulvar-vaginalatrophy are needed.

Vulvovaginal atrophy occurs most often after menopause, but it can alsodevelop during breast-feeding, as a consequence of breast cancertreatment, or at any other time the women's estrogen productiondeclines. Furthermore, recent evidence indicates that women taking oralcontraceptives (which can cause a decline in the production of certainsex hormones such as testosterone) may also experience vulvovaginalatrophy. (See Sex Med 2012, 9, 2213-2223; Sex Med 2010, 7, 1585-1587).Vulvovaginal atrophy can lead to syndromes described as vulvodynia andvestibulodynia.

Vulvodynia is a pain syndrome that affects the vulvar area and oftenoccurs without an identifiable cause or visible pathology. It refers topain of the vulva unexplained by vulvar or vaginal infection or skindisease. Pain is the most notable symptom of vulvodynia, and can becharacterized as a burning, stinging, irritation or sharp pain thatoccurs in the vulva, including the labia and entrance to the vagina. Itmay be constant, intermittent or happen only when the vulva is touched,but vulvodynia is usually defined as lasting for months to years.Symptoms of vulvodynia may occur in one place or the entire vulvar area.It can occur during or after sexual activity, when tampons are inserted,or when prolonged pressure is applied to the vulva, such as duringsitting, bike riding, or horseback riding. Some cases of vulvodynia areidiopathic where no particular cause can be determined. Vestibulodynia,or simply vulvar vestibulitis is vulvodynia localized to the vestibularregion. It tends to be associated with a highly localized “burning” or“cutting” type of pain. The pain of vulvodynia may extend into theclitoris, which is referred to as clitorodynia. Vestibulodynia is themost common subtype of vulvodynia that affects premenopausal women—thesyndrome has been cited as affecting about 10% to 15% of women seekinggynecological care.

Moreover, tissues from women with vulvodynia have been shown to have asignificant increase in subepithelial heparanase activity what may leadto increased intraepithelial hyperinnervation as compared with healthywomen. (See Int J Gynecol Pathol 2008, 27, 136-141). Heparanase, whichis degranulated from mast cells, is capable of degrading the vestibularstroma and epithelial basement membrane, thus permitting stromalproliferation and intraepithelial extension of nerve fibers. Heparanaseis an enzyme that acts within the extracellular matrix to degradeheparan sulfate. Heparan sulfate glycosaminoglycans (which are otherexamples of extracellular matrix components of skin) are abundantcomponents of basement membranes and cell surfaces where they arepresent associated with specific core-proteins to form proteoglycans,mainly perlecan, glypicans and syndecans. They play many roles such asmodulation of cell proliferation and differentiation, cell-matrixadhesion and assembly. Heparan sulfate content has been also shown to bealtered (decrease) during skin aging. This is mainly the result of anincreased formation and activity of its degrading enzyme, heparanase(e.g., Hpse-1), due to UV-B irradiation. (See J Photochem Photobiol B,2012, 106. 107-112). Heparanase activation and the consequent decreaseof heparan sulfate are associated with wrinkle formation. (SeeExperimental Derm 2010, 19, 965-972).

Therefore, vulvodynia is a condition, disorder and disease of the vulva(which is composed of both keratinized and non-keratinized epithelialtissue) that can be also associated with changes in extracellular matrixcomponents.

Vulvar lichen sclerosus, a chronic inflammatory disease which affectsgenital labial, perineal and perianal areas, is another disorderassociated with changes in extracellular matrix components of the vulva.The histopathology of lichen sclerosus suggests abnormalities inextracellular matrix composition and in particular of proteoglycans.(See J Eur Acad Dermatol Venereol, 2012, 26, 207-212).

Vulvar lichen sclerosus produces significant discomfort andpsychological distress in peri- and post-menopausal women. Other vulvardisorders associated with changes in extracellular matrix componentsinclude vulvar lichen planus, erosive lichen planus, vulvar eczema,vulvar lichen simplex chronicus, ulcers of the vulva, Behcet's disease,and vulvar intraepithelial neoplasia.

Finally, it is also accepted that emotional stress (see Brain BehavImmun 2009, 23, 1089-1095), tobacco smoke (see J Investig Dermatol SympProc 2009, 14, 53-55), air pollution, and certain medications (e.g.,corticosteroids) (see Clin Exp Dermatol 1991, 16, 416-419) have anadverse effect on the skin and lead to changes (e.g., decreasedformation and/or increased degradation) of collagen and otherextracellular matrix components.

Compounds that Stimulate Formation of Extracellular Components

Numerous compounds including peptides that stimulate formation ofextracellular matrix components have been reported in the scientificliterature, issued patents, patent applications, or in othercommunications such as technical brochures from suppliers of thosecompounds. The chapter by F. Gorohhui and H. I. Maibach in the Textbookof Aging (2010, Springer (incorporated by reference)) discloses examplesof such compounds; particularly peptides.

Peptides are preferentially used for the stimulation of the formation ofextracellular matrix components for multiple reasons. They are nottoxic, or less toxic than non-peptidic compounds, and no toxic ornoxious degradation products of peptides are formed. Thus, peptidescommonly have fewer side effects, and can be used for the long termtreatment of conditions, disorders and diseases of skin and mucosa inhumans associated with changes in extracellular matrix components. Di-,tri-, and tetra-peptides are particularly well suited for this purpose,as they have the advantage of not being immunogenic or of being lessimmunogenic, in contrast to larger peptides which can become immunogenicand cause allergic reactions. In addition, di-, tri-, and tetra-peptidesare of a lower molecular weight than larger peptides what enables themto better (i.e., faster, in larger quantities) absorb and penetrateskin, mucous membranes, cell membranes, and/or other physiologicalbarriers.

Furthermore, in contrast to natural extracts such as animal, plant orvegetable extracts, peptides can be obtained of a high purity (e.g., 95%and higher) with defined (e.g., the potential impurities are known andtheir content is within the specifications of the peptide product) andreproducible (e.g., from production batch to production batch)characteristics. Natural extracts cannot be obtained in a reproduciblemanner, frequently contain unknown impurities which vary from batch tobatch, and can contain compounds which are not suitable for topicalapplication since they cause skin irritation or sensitization insusceptible individuals. Also, natural extracts are frequently notstable and cause color and/or odor changes of the composition containingsuch extracts. This makes composition with natural extracts, which areknown to stimulate the formation of extracellular matrix components, notuseful for topical application.

The mechanisms of action of peptides are not always known. Stimulationof the formation of extracellular matrix components may be obtained withpeptides acting, for example, on the skin's extracellular matrix,particularly by promoting the synthesis of molecules, by preventingtheir degradation, and/or by acting on receptors.

For example, patent application WO 2007/146269 discloses tetrapeptidesthat are characterized by the amino acid sequence motif GxxG (SEQ IDNO:2) or PxxP (SEQ ID NO:3), where G (glycine) and P (proline) residuesare maintained and x is a variable amino acid. These disclosed sequencesinduce production of collagen from dermal fibroblasts as assessed by adye-binding method designed for the analysis of soluble collagensreleased into the culture medium by mammalian cells during in vitroculture. While some of the disclosed peptides alone or in combinationinduced the synthesis of soluble collagen, none of the disclosedpeptides induce the synthesis of any specific type of collagen (i.e.,such as collagen I and/or III) or induce the synthesis of otherextracellular matrix components.

Likewise, patent application WO 2009/068351 discloses tetrapeptideshaving the motifs GxxG (SEQ ID NO:2), PxxP (SEQ ID NO:3), or PxxK (SEQID NO:4). Preferred peptides disclosed in this application aretetrapeptides selected from the group of GEPG (SEQ ID NO:5), GPPG (SEQID NO:6), GEKG (SEQ ID NO:1), PGPP (SEQ ID NO:7), and/or PKEK (SEQ IDNO:8); or their N-acyl derivatives. While some of the disclosed peptidesalone or in combination induce the synthesis of collagen I (alpha 1, oralpha 2), fibronectin 1, or hyaluronic acid synthetase 1, none of thedisclosed peptides induce the synthesis of collagens III, hyaluronicacid, or other extracellular matrix components.

In a subsequent clinical study (see Exp Dermatol 2011, 20, 602-604), thetetrapeptide GEKG (SEQ ID NO: 1) was further shown to stimulatehyaluronic acid formation after treatment with an oil-in-water vehiclecontaining 50 ppm GEKG (SEQ ID NO: 1) for sixty days

Another example is the tripeptide Gly-His-Lys (GHK), which is a fragmentof collagen released during proteolysis of collagen, or may be derivedfrom the extracellular matrix binding protein SPARC. Its copper complex(GHK-Cu) was studied for tissue remodeling and was shown to stimulatedifferent components of the extracellular matrix including collagens Iand III, elastin, and some glycosaminoglycans. (See J Biomater SciPolymer Edn 2008, 19, 969-988). An in vivo study comparing GHK withGHK-Cu in rat experimental wounds revealed that GHK had no effect andonly GHK-Cu leads to a statistical significant increase of collagenaccumulation. (See J Clin Invest 1993, 92, 2368-2376). As shown in thisstudy, only low and insignificant amounts of type III collagen wereformed by GHK-Cu as compared to total collagen. Whereas higher doses ofGHK-Cu produce skin irritation (see J Biomater Sci Polymer Edn 2008, 19,969-988), GHK and its derivatives do not significantly stimulatecollagen III formation, or stimulate collagen III to a significantlylesser extent than collagen I.

More recently, it was shown that the copper ion (Cu2+) stimulates bothcollagen I and III, but collagen III to a lesser extent than collagen I.(See Connective Tissue Res 2012, 53, 373-378). Therefore, thestimulation of collagen formation observed with GHK-Cu could originatefrom the copper ion and not from the GHK peptide. Copper is highly toxicand therefore not suitable for topical or subcutaneous uses. In additionto the Cu-GHK, the N-palmitoyl derivative of GHK (N-Palmitoyl-GHK) alsostimulates collagen synthesis. (See Int J Cosmetic Sci 2000, 22,207-218).

Patent FR2802413 discloses that N-Palmitoyl-GHK increases the collagensynthesis by up to 75.3% as judged from 3H-proline-incorporationexperiments using human skin explants. In these studies, the collagenincrease was due to formation of collagens I, IV and VII.

While some of these studies disclosed that GHK peptides and derivativesthereof induce the synthesis of collagens I and III and of a few otherextracellular matrix components, none of the disclosed peptides inducethe synthesis of collagen III more than collagen I. In fact, whileGHK-Cu seems to stimulate some collagen III synthesis, those skilled inthe art will recognize that it may be not suitable for prolonged topicalapplication due to its intolerability and the toxicity related tocopper.

As another example, patent application WO2005/048968 discloses thatselected combinations of tripeptide GHK with tetrapeptideGly-Gln-Pro-Arg (SEQ ID NO:9), or their analogues and derivatives,increase the synthesis of collagens I, IV, fibronectin and hyaluronicacid in vitro in a synergistic manner. However, none of the disclosedpeptides, analogues and derivatives, or combinations thereof induces thesynthesis of collagen III or of collagens V and VII.

Patent application WO 2007/143006 discloses polymeric bio-surfactantsconforming to the formula Acyl-AA-Term where Acyl is an 8- to22-membered carbon chain and AA is a consecutive sequence of four tonine amino acid residues, where at least one, preferably at least two ofthe amino acid residues is charged, and Term is an acid C-terminus or anamide C-terminus. While some of the disclosed polymeric bio-surfactants(alone or in combination) induce the synthesis of collagen I,fibronectin, and elastin, none of the disclosed compounds induces thesynthesis of collagens III, hyaluronic acid, or other extracellularmatrix components.

Moreover, patent application WO2010/082175 discloses specific peptideswhich significantly increase the formation of collagen I, III, IV,fibronectin, hyaluronic acid, and laminins. The peptidepalmitoyl-KMO2K—OH increased the collagen I formation by 111% (asdetermined by ELISA) and the collagen III formation by 104% (asdetermined by immunofluorescence) in normal human dermal fibroblasts.Some of the disclosed peptides induce the synthesis of both collagen Iand collagen III. However, none of the disclosed peptides induce thesynthesis of collagen III more than collagen I or induce the synthesisof collagens V and VII, and other extracellular matrix components. Infact, also disclosed in WO2010/082175, the addition of a specific plantextract, Portulaca pilosa, to palmitoyl-KMO2K—OH is required to inducethe synthesis of collagen III more than collagen I by the combination ina synergistic manner.

WO2010/136965 discloses dipeptides of the formula R1-Tyr-Arg-R2 whichsignificantly increase the formation of elastin/tropelastin, fibrillin1, and decorin in human dermal fibroblasts. However, none of thedisclosed peptides, analogues and derivatives, or combinations thereofinduces the synthesis of collagens, or other extracellular matrixcomponents.

Other examples known in the art are tri-peptides with the formula KxK,which have been proposed as TGF-beta growth factor activators therebyenabling the synthesis of collagen in the skin extracellular matrix. Forexample, the tripeptide proposed in FR2810323 is elaidyl-KFK. However,in patent EP1625150 the inventors describe elaidyl-KFK as beinginsufficiently active to stimulate collagen synthesis and propose toalter the lysine side chains, for example the length of the aminatedalkyl chains or to introduce specific side chains or to use a centralamino acid X with a hydrocarbon chain possibly substituted by a hydroxylgroup. Among the examples given are palmitoyl-KVK, palmitoyl-KAK orpalmitoyl-KSK.

Also known in the art are peptides with trade names Kollaren® (INCIname: tripeptide-1), Collaxyl® IS (INCI name: hexapeptide-9), SYN®-TC(combination of palmitoyl-Lys-Val-Lys-OH with palmitoyl-Lys-Val-Thr-OHand tetradecylaminocarbonyl-Dab-Val-Dab) that significantly increase theformation of both collagens I and III in human dermal fibroblasts.However, none of these disclosed peptides, analogues and derivatives, orcombinations thereof induce the synthesis of collagen III to a higherdegree than the synthesis of collagen I. In addition, those otherpeptides are limited to the stimulation of only a few additionalextracellular matrix components, most of which are not associated withskin aging. Moreover, these peptides either do not stimulate, onlyrelatively weakly stimulate, and/or only partially stimulate theformation of the extracellular matrix components that have beendescribed to be significantly altered with skin aging, namely collagenI, collagen III, collagen V, elastin, and hyaluronic acid. Moreovercollagen III formation is either not stimulated by the peptides of theprior art, or, it is stimulated by the peptides to a lesser degree thancollagen I formation. Thus, in contrast to the compositions of theinstant invention, these peptides stimulate collagen I formationsignificantly more than collagen III formation.

Collagen III is an important component of the extracellular matrix. Boththe formation and structure of collagen III are changed with aging,wounding or damaging of skin, as well as many conditions, disorders anddiseases of skin and mucosa associated with changes in extracellularmatrix components. Therefore, there is a need for compositions thateffectively stimulate collagen III formation in skin and mucosa; forcompositions that effectively stimulate collagen III withoutsignificantly stimulating collagen I formation; and/or for compositionsthat effectively stimulate collagen III significantly more thanstimulating collagen I formation. Such compositions are particularlyuseful for scarless repair after wounding of skin as well as for optimalrestoration of damaged skin.

More particularly, there is a need for compositions which stimulatecollagen I, collagen III, collagen V, elastin and/or hyaluronic acid(e.g., all of the extracellular matrix components that are thought to besignificantly altered with skin aging). There is also a need forcompositions which stimulate extracellular matrix components such asfibrillins, which help in the formation of elastic fibers or otherextracellular matrix components.

Preferably, the compositions of the present invention contain OctanoylCarnosine (or one or more derivatives thereof). The compositions of thepresent invention include Octanoyl Carnosine (or one or more derivativesthereof) at concentration sufficient for demonstrating clinical efficacyfor improving skin aging, or other skin disorders and diseasesassociated with skin aging.

The compositions of the present invention incorporate Octanoyl Carnosine(or one or more derivatives thereof) at concentration sufficient fordemonstrating clinical efficacy for wound healing of skin, and/or forenhancing the restoration of skin after cosmetic and dermatologicalprocedures. The compositions of the present invention can incorporateOctanoyl Carnosine (or one or more derivatives thereof) at concentrationsufficient for demonstrating clinical efficacy for scarless woundhealing of wounded skin.

In another example, the compositions of the present inventionincorporate Octanoyl Carnosine (or one or more derivatives thereof) atconcentration sufficient for demonstrating clinical efficacy for thetreatment of aging vulvar and vaginal tissue.

In another example, the compositions of the present inventionincorporate Octanoyl Carnosine (or one or more derivatives thereof) atconcentration sufficient for demonstrating clinical efficacy fortreating symptoms, disorders and diseases of the vulvar and the vaginaltissue which can be associated with vulvovaginal atrophy.

In yet another example, the compositions of the present inventionincorporate Octanoyl Carnosine (or one or more derivatives thereof) atconcentration sufficient for demonstrating clinical efficacy fortreating vulvodynia.

In yet another example, the compositions of the present inventionincorporate Octanoyl Carnosine (or one or more derivatives thereof) atconcentration sufficient for demonstrating clinical efficacy fortreating lichen sclerosus.

The compositions of the present invention incorporate Octanoyl Carnosine(or one or more derivatives thereof) at concentration sufficient fordemonstrating clinical efficacy for treating other conditions, disordersand diseases of skin and mucosa in humans associated with changes inextracellular matrix components, including but not limited to thetreatment of atrophy of any human tissue.

For example, the compositions of the invention contain OctanoylCarnosine (or one or more derivatives thereof) at a concentrationbetween 0.0001% to 10% Octanoyl Carnosine (or one or more derivativesthereof) per weight; depending on the solubility of Octanoyl Carnosine(or one or more derivatives thereof) in the composition.

The compositions may also contain selected tri-peptides (e.g.,Palmitoyl-GHK) (or one or more derivatives thereof) and/or selectedtetra-peptides (e.g., GEKG (SEQ ID NO:1)) (or one or more derivativesthereof). Preferably, the composition contains Octanoyl Carnosine,Palmitoyl-GHK, and GEKG (SEQ ID NO: 1) (or one or more derivativesthereof) at a weight ratio of about 4:1:5. However, determination ofother suitable weight ratios between these components is within theroutine level of skill in the art.

When combining Octanoyl Carnosine (or one or more derivatives thereof)with both Palmitoyl-GHK (or one or more derivatives thereof) and GEKG(SEQ ID NO: 1) (or one or more derivatives thereof), the optimal weightratio of Octanoyl Carnosine (or one or more derivatives thereof) toPalmitoyl-GHK (or one or more derivatives thereof) to GEKG (SEQ IDNO: 1) (or one or more derivatives thereof) is 4 parts OctanoylCarnosine (or one or more derivatives thereof):1 part Palmitoyl-GHK (orone or more derivatives thereof):5 parts GEKG (SEQ ID NO: 1) (or one ormore derivatives thereof) (parts refer to parts per weight), asdiscovered during in vitro studies for hyaluronic acid formation (seeExample 5, infra). Thus, one of the preferred compositions containsOctanoyl Carnosine (or one or more derivatives thereof), Palmitoyl-GHK(or one or more derivatives thereof) and GEKG (SEQ ID NO:1) (or one ormore derivatives thereof) at a weight ratio of 4:1:5; whereas OctanoylCarnosine is present in the compositions in proportions between 0.001%and 1%, in a carrier or excipient acceptable for topical application.

The combination of Octanoyl Carnosine, Palmitoyl-GHK and GEKG (SEQ IDNO: 1) (or one or more derivatives thereof) at the weight ratio of 4:1:5is not known in the art. The combination of Octanoyl Carnosine,Palmitoyl-GHK and GEKG (SEQ ID NO: 1) (or one or more derivativesthereof) at the weight ratio of 4:1:5 stimulates the synthesis ofhyaluronic acid at this specific weight ratio in synergistic andunexpected manner.

One or more additional ingredients, including one or more additionalsubstances (e.g., acceptable carriers and/or excipients) suitable fortopical application can also preferably be used in these compositions.The one or more additional ingredients may also include additionalsubstances with biological activities (i.e., biologically activeagents).

Also provided are methods for improving the state and appearance ofhuman skin and reducing the signs of skin aging by using a compositioncontaining Octanoyl Carnosine (or one or more derivatives thereof), orOctanoyl Carnosine (or one or more derivatives thereof) in combinationwith one or more additional selected tri- and/or tetra-peptides (or oneor more derivatives thereof) according to the present invention, thatstimulate the formation of extracellular matrix components.

The invention additionally provides methods of maintaining healthy skinand/or preventing skin aging by using a composition containing OctanoylCarnosine (or one or more derivatives thereof), or Octanoyl Carnosine(or one or more derivatives thereof) in combination with selected tri-and/or tetra-peptides according to the present invention, that stimulatethe formation of extracellular matrix components.

Additionally, the invention also provides methods of enhancing therestoration of skin after cosmetic and dermatological procedures,enhancing wound healing, reducing the atrophy of any human tissueincluding vulvovaginal atrophy, and improving other conditions,disorders and diseases of skin and mucosa in humans associated withchanges in extracellular matrix components by using a compositioncontaining Octanoyl Carnosine (or one or more derivatives thereof), orOctanoyl Carnosine (or one or more derivatives thereof) in combinationwith selected tri- and/or tetra-peptides according to the presentinvention, that stimulate the formation of extracellular matrixcomponents.

In any of the method described herein, skin or skin cells (e.g.,epidermal keratinocytes, dermal fibroblasts) are contacted (i.e.,topically, subcutaneously, or by any other suitable method known in theart) with a composition containing Octanoyl Carnosine (or one or morederivatives thereof), or Octanoyl Carnosine (or one or more derivativesthereof) in combination with selected tri- and/or tetra-peptides.Additionally, the methods may also involve contacting (i.e., topically,subcutaneously, or by any other suitable method known in the art) mucosa(i.e., mucous membranes) or mucosal cells (i.e., epithelial cells) witha composition containing Octanoyl Carnosine (or one or more derivativesthereof), or Octanoyl Carnosine (or one or more derivatives thereof) incombination with selected tri- and/or tetra-peptides according to thepresent invention.

The compositions can be an aerosol, emulsion, liquid, lotion, cream,paste, ointment, foam, patch, microneedle device or any other cosmetic,dermatological and pharmaceutically acceptable formulation or device.Generally, an acceptable formulation for cosmetic, dermatological,and/or pharmaceutically use would include any acceptable carrier,excipient, and/or substance suitable for use on human skin or mucosa.The compositions may also contain one or more other biologically activeagents including, but not limited to, retinoids, growth factors, and/orother peptides.

Any of the compositions of the present invention may also be used incombination with other cosmetic, skin care, feminine, hygiene,dermatological, pharmaceutical products, and/or medical devices.

The invention also provides methods of reducing scarring of skin damagedby normal aging, disease, injury, trauma, or by surgery or other medicalprocedures. Such methods can involve administering to the wound of ahuman a composition, wherein the composition contains any of theabove-described peptides, singularly or in combination. The compositionsmay also be used in combination with other therapeutic agents, forexample, such as tissue grafts, tissue culture products, oxygen ordressings.

The compositions of the invention can be used in humans. Alternatively,the composition may also be used in any kind of animal, preferably inmammals, and more preferably in cows, horses, cats, dogs, pigs, goats,or sheep.

Carnosine

As used herein, the term “carnosine” includes and encompasses thedi-peptides beta-alanyl-histidine (see Formula II, supra) and allrelated compounds such as anserine (beta-alanyl-1-methyl-histidine) andhomocarnosine (gamma-amino-butyryl-histidine). As used herein the term“carnosine” also includes D, L-carnosine, D-carnosine, L-carnosine, aswell as salts thereof.

Carnosine's properties, functions and potential therapeutic applicationshave been extensively reviewed. (See P. J. Quinn et al. (Molec AspectsMed 1992, 13, 379-444) (incorporated herein by reference)). Althoughcarnosine's anti-aging properties have previously been described, theproperties of Octanoyl Carnosine have not previously been described andare surprising and unexpected. As reviewed by A. R. Hipkiss (Int JBiochem Cell Biol 1998, 30, 863-868; Cell Mol Life Sci 2000, 57,747-753; Experimental Gerontology 2009, 44, 237-242 (incorporated hereinby reference)), the naturally occurring dipeptidebeta-alanyl-L-histidine (L-carnosine) is found in large amounts inlong-lived tissues. It has protective functions in addition to itsanti-oxidant and free-radical scavenging roles, and it extends culturedhuman fibroblast life-span, kills transformed cells, protects cellsagainst aldehydes and an amyloid peptide fragment, and acts as ananti-glycating agent. More recently, carnosine was shown to protectagainst telomere shortening in cultured human fibroblasts and to extendthe life-span of senescence-accelerated mice and Drosophila flies.Studies have revealed carnosine-induced upregulation of stress proteinexpression and nitric oxide synthesis, both of which may stimulateproteasomal elimination of altered proteins. As carnosine exertsanti-convulsant effects in rodents, the dipeptide may participate in therepair of protein isoaspartyl groups.

Carnosine has also been shown to promote healing of skin wounds, gastricand duodenal ulcers, as well as corneal and pulmonary wounds. (SeeNutrition 1998, 14, 266-269; Molec Aspects Med 1992, 13, 379-444). Forinstance, it has been shown to accelerate healing of bleomycin-inducedand irradiation-induced pulmonary wounds. (See Am J Physiol Lung CellMol Physiol 2007, 292, L1095-L1104; Med Hypotheses 2006, 66, 957-959).These effects of carnosine have been attributed to its affinity toquench free radicals. In dermal wounds, carnosine promotes granulation,increases the tensile strength and hydroxyproline content in the woundarea. (See Surgery 91:56-60 (1982); Surgery 100:815-21 (1986)). Theobserved effect has been ascribed to the histamine synthesis fromhistidine, one of the components of carnosine and the stimulation ofcollagen synthesis by beta-alanine, another component of carnosine.

Other investigations on the role of carnosine in the wound healing ofsurgical wounds have shown that carnosine is enhancing biosynthesis ofglycosaminoglycans. (See Surgery 91:56-60 (1982); Cell Mol Biol Inc CytoEnzymol 23:267-73 (1978); Cell Mol Biol 29:1-9 (1983)). Nagai et al.(Surgery 100: 815-821 (1986)) explain the mechanisms of action ofcarnosine in wound healing as beta-alanine induced collagen synthesiscombined with histamine formation, which promotes granulation andresults in a faster healing of the wound tissue.

In a more recent study of diabetic wound healing in a diabetic animalmodel (see Amino Acids 2012, 43:127-134), it was observed that dailyinjections and local application of carnosine significantly enhanced theexpression of extracellular matrix components collagen I and smoothmuscle actin, as well as certain growth factors. Carnosine treatmentcaused a significant increase in IGF1 expression in the wound areasuggesting a direct or indirect modulation of IGF1 expression bycarnosine enabling improved wound healing. Similarly, TGF-beta, which iswell studied for its role in cutaneous wounds in various phases of woundhealing, is significantly over-expressed in the wound tissue fromcarnosine-treated.

Apart from these effects, the wound healing potential of carnosine mayalso be due to its ability to alter the generation of free radicals andoxidative stress-induced prolonging of the inflammatory phase of thewound healing process in a diabetic wound. Since carnosine also shiftsthe acid-base balance to higher pH values, it can modify the woundmicro-environment and block the activity of acid proteases and therebyenhance wound healing under hyperglycemic conditions.

In another recent study (see Neuro Endocrinol Lett 2010, 31 Suppl 2:96-100), carnosine was shown to inhibit the degradation of hyaluronaninduced by free radicals (hydroxy and peroxy-type radicals) in vitro.Importantly, as demonstrated in this study, and in contrast to thefindings of the present invention using Octanoyl Carnosine (or one ormore derivatives thereof) and combinations of Octanoyl Carnosine (or oneor more derivatives thereof) with selected tri- and/or tetra-peptides,carnosine does not induce the formation of hyaluronan but, rather,limits its degradation through carnosine's antioxidant properties. Thus,the increase in glycosaminoglycans observed in earlier studies (see,e.g., Surgery 91:56-60 (1982); Cell Mol Biol Incl Cyto Enzymol 23:267-73(1978); Cell Mol Biol 29:1-9 (1983)) is the result of a decreaseddegradation and not of an enhanced formation of glycosaminoglycans.

Carnosine was also shown to inhibit high-glucose-mediated matrixaccumulation in human mesangial cells by interfering with TGF-βproduction and signaling. (See Nephrol Dial Transplant 2011, 26,3852-3858). Under high-glucose conditions, deposition of collagen VI andfibronectin were increased, what was significantly inhibited on theprotein and messenger RNA level by carnosine. TGF-β production increasedunder high-glucose conditions but was completely normalized by additionof L-carnosine.

Moreover, in a study investigating liver disorders induced bySchistosoma mansoni parasite in hamsters, subcutaneous administration ofcarnosine lowered the serum pro-collagen III peptide level in infestedhamsters. (See Comp Biochem Physiol B Biochem Mol Biol 2002, 131,535-542).

In diabetic nephropathy, carnosine inhibited the increased formation offibronectin and collagen type VI in podocytes and the increasedproduction of TGF-beta in mesangial cells induced by glucose. (SeeDiabetes 2005, 54, 2320-2327). Thus, carnosine protects against theadverse effects of high glucose levels on renal cells.

Therefore, all these effects (including carnosine's wound healingproperties) can be attributed to carnosine's anti-oxidant properties,anti-glycation effects (in particular under hyperglycemic conditions),as well as the histamine synthesis from histidine as a component ofcarnosine, and/or stimulation of collagen I synthesis by beta-alanine,which is another component of carnosine.

In summary, carnosine has not been described to induce the formation ofcollagen III to a higher degree than collagen I, or to stimulate theformation of all extracellular matrix components that have beendescribed to be significantly altered with skin aging, namely collagenI, collagen III, collagen V, elastin, and hyaluronic acid.

Octanoyl Carnosine and Other N-Acylated Derivatives of Carnosine

The term “N-Octanoyl Carnosine” (also referred to interchangeably hereinas “Octanoyl Carnosine”) in accordance with the present inventionincludes and encompasses all derivatives of carnosine where theamino-terminus of the di-peptide is acylated to form an octanoyl group.As used herein the term “Octanoyl Carnosine” includes octanoylD,L-carnosine, octanoyl D-carnosine, octanoyl L-carnosine; as well astheir salts. The terms Octanoyl Carnosine and N-Octanoyl Carnosine aresynonyms that are used interchangeably herein. Octanoyl Carnosine can beproduced by reaction of carnosine with octanoic acid (caprylic acid)under appropriate conditions. (See Example 1, infra).

Octanoyl Carnosine is an example of an N-acylated derivative ofcarnosine. N-Acylated derivatives of carnosine can be obtained afterreacting the free amino-group (N-terminal amino group) of carnosine withan acylating agent under appropriate conditions known in the art ofpeptide synthesis. The acylating agent can be activated, using generaltechniques known in the art such as those described for example in“Amide bond formation and peptide coupling” (Tetrahedron 61(46),10827-10852, 2005). Examples of activated acylating agents include, butare not limited to, acid chlorides, acid bromides, acid fluorides,symmetrical anhydrides, mixed anhydrides, carboxylic acids activatedusing common carbodiimides such as, but not limited to,diisopropylcarbodiimide (DIPCDI), N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3′-dimethylamino-propyl)carbodiimide hydrochloride (EDC).Other non-limiting examples include carboxylic acids using theaforementioned carbodiimides and an additive, including, but not limitedto, N-hydroxysuccinimide (HOSu), N-hydroxybenzotriazol (HOBt),1-Hydroxy-7-azabenzotriazol, 6-chloro-N-hydroxybenzotriazol (HOAt),3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (DhbtOH) orp-nitrophenol (PNP). Other examples include, but are not limited to,carboxylic acids activated with an uronium salt or a phosphonium salt,such as but not limited to, O-Benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU),O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU), 2-(6-Chloro-1H-benzotriazole-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU),2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate (TCTU),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),2-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HDBTU), 2-Succinimido-1,1,3,3-tetramethyluroniumhexafluorophosphate (HSTU),N,N,N′,N′-Tetramethyl-O-(succinimidyl)uronium tetrafluoroborate (TSTU),2-(endo-5-norbornene-2,3-dicarboxymido)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HNTU),1-benzotriazolyoxytris-(dimethylamino)phosphonium hexafluorophosphate(BOP) or benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PYBOP).

Other activated acylating agents include, but are not limited to, estersof N-hydroxysuccinimide (NHS ester), p-nitrophenol (PNP ester),N-Hydroxy-5-norbornene-2,3-dicarboxylimide (HONB-ester),N-pentafluorophenol ester (PfP-ester), 2,4-dinitrophenyl ester,4-nitrophenyl ester, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine(HODhbt), carbonyldiimidazole (CDI) orN-ethyl-5-phenylisoxazolium-3′-sulfonate (NEPIS), preferably aN-hydroxysuccinimide ester, p-nitrophenol or a HOBt ester or aderivative thereof using reaction conditions as, e.g., described inOrganic Synthesis on solid Phase (Florencio Zaragoza Dorwald, Wiley-VCHVerlag GmbH, D-69469 Weinheim, 2000), Novabiochem Catalog (MerckBiosciences 2006/2007) and Fmoc Solid Phase Peptide Synthesis (Edited byW. C. Chan and P. D. White, Oxford University Press, 2000, ISBN0-19-963724-5).

As known in the art, the same (or similar) methods of obtaining orproducing N-acylated derivatives of carnosine can also be used to obtainN-acylated derivatives of other peptides, including but not limited to,the tri-peptide GHK, GHK esters, the tetra-peptide GEKG (SEQ ID NO:1),and GEKG (SEQ ID NO:1) esters.

N-Acetyl-Carnosine

The N-acetylated derivative of carnosine, N-acetylcarnosine, haspreviously been described for uses in ophthalmology. (See Recent PatDrug Deliv Formul 2009, 3, 229-65). For instance, patent applications WO2004/028536 A1; WO 94/19325; WO 95/12581; WO 2004/064866 A1 describeN-acetylcarnosine lubricant eye drops designed as 1% N-acetylcarnosineprodrug of L-carnosine containing a mucoadhesive cellulose-basedcompound combined with corneal absorption promoters in a drug deliverysystem for the management of age-related serious or disabling eyediseases in humans (age-related cataracts, ocular inflammation,age-related macular degeneration, macular dystrophies, ocularmanifestations of diabetes, hypertonic retinopathy, primary open angleglaucoma, vitreous lesions).

Acetylation makes N-acetylcarnosine more resistant to degradation bycarnosinase, an enzyme that breaks down carnosine to its constituentamino acids, beta-alanine and histidine. This results in theprolongation and potentiation of physiological responses to treatmentswith carnosine as antioxidant and anti-glycation agent. (See Life Sci2006, 11, 78, 2343-57). For the prolongation and potentiation of theantioxidant protective effects to the cosmetic, therapeutic and medicaltreatments with carnosine, N-acetylcarnosine has also been proposed forthe use in skin care products. (See Life Sci 2006, 78, 2343-2357).

As is true for N-acetylcarnosine, Octanoyl Carnosine is also highlyresistant to hydrolysis by carnosinases and provides the antioxidantprotective and anti-glycation effects characteristic for carnosine for alonger period of time as compared to unmodified carnosine.

N-acetylcarnosine has not been described to induce the formation ofcollagen III to a higher degree than collagen I or to stimulate theformation of all extracellular matrix components that have beendescribed to be significantly altered with skin aging, namely collagenI, collagen III, collagen V, elastin, and hyaluronic acid.

Esters of Carnosine

Esters of carnosine include ester derivatives of carnosine which can beobtained after reacting the free carboxyl-group (free acid C-terminus)of carnosine with an esterifying agent under appropriate conditionsknown in the art of peptide synthesis. In one non-limiting method, anacidified alcohol solution is used for the esterification of carnosine.The method includes, for example, the use of an acidified alcohol suchas methanolic HCl to generate a carnosine ester, e.g., carnosine methylester. Additionally, other alcohols (such as, for example, ethanol,propanol, or isopropanol) or substituted alcohols (such as, for example,aminoethanol) can be used to generate a reagent for esterification ofthe free acid C-terminus of carnosine.

Suitable alcohols for esterification of carnosine include but are notlimited to organic chemicals with at least one hydroxyl group such asalkyl alcohols (e.g., C1 to C20 primary alcohols, C3 to C20 secondaryalcohols), aryl alcohols (e.g.: benzyl alcohol), or poly-alcohols suchas sugar alcohols (e.g., mono-, di-, poly-saccharides) or glycols andpoly-glycols (e.g., glycerin, ethylene glycol, propylene glycol, PEGs,PPGs). Ascorbic acid is also a suitable agent (since it has at least onefree hydroxyl group) useful for esterification with carnosine.

Any methods to obtain esters of carnosine known in the art can also beused to obtain esters of other peptides (including the esterification offree carboxyl groups of the peptides other than the free acidC-terminus) including, but not limited to, peptides GEKG (SEQ ID NO:1),N-Acyl-GEKG (SEQ ID NO:1), and/or N-Palmitoyl-GHK.

For example, PEGylation of carnosine, Octanoyl Carnosine,N-Palmitoyl-GHK,

GEKG (SEQ ID NO: 1), and/or N-Acyl-GEKG (SEQ ID NO: 1) is one preferredesterification method to prolong half-life, increase stability andprovide higher water solubility of those peptides.

Peptides

The term “peptide” in accordance with the present invention is acompound that includes an uninterrupted sequence of at least two aminoacids within its structure. The terms “di-peptide” or “dipeptide” asused herein refer to a compound that includes an uninterrupted sequenceof two amino acids within its structure. The terms “tri-peptide” or“tripeptide” as used herein refer to a compound that includes anuninterrupted sequence of three amino acids within its structure. Asused herein, a “tetra-peptide” or “tetrapeptide” is a compound thatincludes an uninterrupted sequence of four amino acids within itsstructure. These amino acids are indicated herein using a traditionalone letter convention from left (N-terminal end) to right (C-terminalend). In this nomenclature, G is glycine, H is histidine, K is lysine,and E is glutamic acid.

The term “amino acid” as used herein includes and encompasses all of thenaturally occurring amino acids, either in the D- or L-configuration ifoptically active, and the known non-native, synthetic, and modifiedamino acids, such as homocysteine, ornithine, norleucine and p-valine. Alist of non-natural amino acids may be found in The Peptides, Vol. 5(1983), Academic Press, Chapter VI, by D. C. Roberts and F. Vellaccio(incorporated herein by reference). The amino acids in the peptides ofthe present invention may be present in their natural L-configuration,unnatural D-configuration, or as a racemic mixture.

As used herein, the term “peptide” shall also refer to salts,deprotected forms, acylated forms of the peptide, deacylated forms ofthe peptide, enantiomers, diastereomers, racemates, prodrugs andhydrates of the above-mentioned peptide. Diastereomers of the peptideare obtained when the stereochemical or chiral center of one or moreamino acids is changed. The enantiomer has the opposite stereochemistryat all chiral centers.

The term “prodrug” refers to any precursor compound which is able togenerate or to release the above-mentioned peptide under physiologicalconditions. Such prodrugs are for instance larger peptides which areselectively cleaved in order to form the peptide of the invention.Further prodrugs are protected amino acids having especially protectinggroups at the carboxylic acid and/or amino group. Suitable protectinggroups for amino groups are the benzyloxycarbonyl, t-butyloxycarbonyl(BOC), formyl, and acetyl or acyl group. Suitable protecting groups forthe carboxylic acid group are esters such as benzyl esters or t-butylesters.

Peptides are synthesized by coupling the carboxyl group or C-terminus ofone amino acid to the amino group or N-terminus of another. Due to thepossibility of unintended reactions, protecting groups are usuallynecessary. Chemical peptide synthesis starts at the C-terminal end ofthe peptide and ends at the N-terminus. Peptides can be synthesizedeither by solid-phase peptide synthesis, by liquid-phase peptidesynthesis, or by fragment condensation. In principle, the seeminglysimple formation of a peptide bond can be accomplished using all theprocedures available in organic chemistry for the synthesis ofcarboxylic acid amides. However, due to the presence of variousfunctional groups in natural and unnatural amino acids and particularlythe requirement for full retention of chiral integrity, the coupling ofamino acids and peptides under mild conditions can be challenging. Aplethora of coupling reagents has been developed suitability forspecific applications (e.g., solid-phase peptide synthesis or fragmentcondensation). All coupling methods have the same reaction principle incommon: after activation of the carboxyl group of the first amino acid,the second amino acid can form the peptide bond by a nucleophilic attackof its amino group. In order to prevent uncontrolled peptide bondformation the amino group of the first amino acid and all functionalside chain groups need to be reversibly blocked. Repeated de-blocking,activation, and coupling build the peptide to its desired finalsequence.

The general process for synthesizing peptides on solid-phase (e.g.,resin) starts by attaching the first amino acid, the C-terminal residue,to the resin. To prevent the polymerization of the amino acid, the alphaamino group and the reactive side chains are protected with a temporaryprotecting group. Once the amino acid is attached to the resin, theresin is filtered and washed to remove byproducts and excess reagents.Next, the N-alpha protecting group is removed in a deprotection processand the resin is again washed to remove byproducts and excess reagents.Then the next amino acid is coupled to the attached amino acid. This isfollowed by another washing procedure, which leaves the resin-peptideready for the next coupling cycle. The cycle is repeated until thepeptide sequence is complete. Then typically, all the protecting groupsare removed and the peptide resin is washed, and the peptide is cleavedfrom the resin.

The invention is further directed towards a method for producing thedisclosed peptides. The peptides may be produced using any method knownto those skilled in the art such as those disclosed in Merrifield, R.B., Solid Phase Peptide Synthesis I., J. AM. CHEM. SOC. 85:2149-2154(1963); Carpino, L. A. et al., [(9-Fluorenylmethyl)Oxy]Carbonyl (Fmoc)Amino Acid Chlorides: Synthesis, Characterization, And Application ToThe Rapid Synthesis Of Short Peptides, J. ORG. CHEM. 37:51:3732-3734;Merrifield, R. B. et al., Instrument For Automated Synthesis OfPeptides, ANAL. CHEM. 38:1905-1914 (1966); or Kent, S. B. H. et al.,High Yield Chemical Synthesis Of Biologically Active Peptides On AnAutomated Peptide Synthesizer Of Novel Design, IN: PEPTIDES 1984(Ragnarsson U., ed.) Almqvist and Wiksell Int., Stockholm (Sweden), pp.185-188, all of which are incorporated by reference herein in theirentirety. Preferably, the peptides are manufactured by solid-phasesynthesis. However, the peptides may also be manufactured using standardsolution phase methodology.

Preferably, the peptides are in their trifluoroacetate (TCA) and/oracetate salt forms. However, the peptides can be in any other salt formincluding, but not limited to, adipate, ascorbate, alginate, benzoate,benzenesulfonate, bromide, carbonate, citrate, chloride, dibutylphosphate, dihydrogen citrate, dioctyl phosphate, dihexadecyl phosphate,fumarate, gluconate, glucuronate, glutamate, hydrogen carbonate,hydrogen tartrate, hydro-chloride, hydrogen citrate, iodide, lactate,liponic acid, malate maleate, malonate, palmoate (embonate), palmitate,phosphate, salicylate, stearate, succinate, sulfate, tartrate, tannate,oleate, octyl phosphate; any other salts of the phosphate or carboxylatefamily; and/or any combination thereof. Under certain conditions, othersalts can be derived from inorganic bases, include by way of exampleonly, sodium, potassium, lithium, ammonium, calcium and magnesium salts.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary and tertiary amines.

The term “peptide”, as used herein, includes proteins.

Skin Aging

“Signs of skin aging” include, but are not limited to, all outwardvisibly and tactilely perceptible manifestations as well as any othermacro or micro effects due to skin aging. Such signs may be induced orcaused by intrinsic factors (showing as chronological aged skin) andextrinsic factors (showing as environmental skin damage including butnot limited photo-aged skin). These signs may result from processeswhich include, but are not limited to, the development of texturaldiscontinuities such as wrinkles and coarse deep wrinkles, fine or skinlines, crevices, bumps, large pores (e.g., associated with adnexalstructures such as sweat gland ducts, sebaceous glands, or hairfollicles), or unevenness or roughness, loss of skin elasticity (lossand/or inactivation of functional skin elastin), sagging (includingpuffiness in the eye area and jowls), loss of skin firmness, loss ofskin tightness, loss of skin recoil from deformation, discoloration(including under eye circles), blotching, sallowness, hyperpigmentedskin regions such as age spots and freckles, keratoses, abnormaldifferentiation, hyperkeratinization, elastosis, collagen breakdown, andother histological changes in the stratum corneum, dermis, epidermis,the skin vascular system (e.g., telangiectasia or spider vessels), andunderlying tissues, especially those proximate to the skin.

As used herein, prophylactically regulating a skin condition includesdelaying, minimizing and/or preventing visible and/or tactilediscontinuities in skin (e.g., texture irregularities in the skin whichmay be detected visually or by feel), including signs of skin aging. Asused herein, therapeutically regulating skin condition includesameliorating, e.g., diminishing, minimizing and/or effacing,discontinuities in skin, including signs of skin aging. Some of thecompositions of the present invention may be used for prophylacticallyand/or therapeutically regulating a skin condition.

Some of the compositions of the present invention are useful forimproving skin appearance and/or feel. For example, some preferredcompositions of the present invention are useful for regulating theappearance of skin condition by providing an immediate visualimprovement in skin appearance following application of the compositionto the skin. Generally speaking, compositions of the present inventionwhich further contain particulate materials will be most useful forproviding the immediate visual improvement.

Demonstrating Clinical Efficacy

Prevention, amelioration, and/or treating of the signs of skin aging,protection and/or improving skin condition, and the prevention and/ortreatment of skin imperfections are functional features which can bevisualized, analyzed, measured and quantified using many techniquesknown by the specialist in cosmetic or skin rejuvenation treatments.Decrease of fine lines, wrinkles, skin folds, and of skin roughness canbe quantified either directly on the person contact-free using fringeprojection (FOITS=Fast Optical In vivo Topometry System; Dermatop™ orPrimos™ system), or by silicon replicas of the skin area which are thenanalyzed by the technique called “drop shadows” or by a FOITS system, orby a Canfield VISIA™ device. Changes in volume and shape of the face canbe quantified using a relief obtaining system without contact using afringe projection FOITS system. Alteration of the skin barrier can bequantified by measuring transepidermal water loss (TEWL) using aTewameter™, a Vapometer™, a Dermalab™, and/or an Aquaflux™ device. Lossof firmness and/or elasticity and/or tone and fatigue of the skin can bequantified using a Cutometer™, a Reviscometer™, an Aeroflexmeter™, aDynaskin™, a Ballistometer™, a Twistometer™ and/or a Dermalab™ device.Dull complexion, loss of uniformity of skin tone, pigmentation changes(hypo and hyper pigmentation), local reddening, loss of clarity andbrightness of the complexion, pigmentation spots, rosacea, dark circlesare directly measurable using a Mexameter™, a Chromameter™, aColormeter™, a Canfield VISIA™, a Canfield VISIA-CR™, a SIAscope™, aGoniolux™ or a confocal laser microscope device, and/or by specificcolor analysis on photo (enabled by the technique of photographing inpolarized crossed and parallel light). The number and size of facialpores can be quantified by the silicon replica technology describedabove, or by specific analysis on photo (enabled by using a videomicroscope or a macroscopic photographing system). Atrophy and thinningof the skin, epidermis, dermis, or hypodermis (e.g., in case of studyingslimming agents) is measurable by measuring TEWL (e.g., in case ofstudying the epidermis), or by an ultrasound echographic device, and/ora confocal laser microscope device. Density of skin fibers can bequantified by ultrasound and then by image analysis. Cellulite isquantified either directly by a relief obtaining system without contactusing fringe projection (FOITS) or indirectly by measuring the length ofthe dermo-hypodermal junction by an ultrasound echographic device.Stretch marks are either directly quantified using a relief obtainingsystem without contact using fringe projection (FOITS) or by the siliconreplica technology. Skin softness is directly measurable by techniquesof friction study as with a frictiometer device or indirectly by thesilicon replica technology. Changes in collagen, extracellular matrixcomponents, and/or in connective tissue fibers may be quantified byhistology, confocal laser microscopy, UV spectroscopy, SIAscopie, and/orby multiphoton spectroscopy. All changes visible to the eye (includingbut not limited to fine lines, wrinkles, folds, texture, sagging, lossof elasticity color, tone, pigmentation, redness) can be quantified indirect or on photography, by a trained judge person or not, with orwithout visual scoring system (e.g., using a 4-point severity scale).

Cosmetic Composition and Medicament

The terms “cosmetic composition” and “cosmetic product” are usedinterchangeably herein relate formulations that can be used for cosmeticpurposes or purposes of hygiene or as a basis for delivery of one ormore cosmetic and/or pharmaceutical substances, products, and/oringredients.

The terms “pharmaceutical composition” and “medicament” is used hereinto refer to a formulation that can be used for medical purposes or as abasis for delivery of one or more cosmetic and/or pharmaceuticalsubstances, products, and/or ingredients.

It is possible that any of the formulations, compositions, medicaments,and/or products described herein can be used for two or more of thesesame purposes at one time.

Preferably, the compositions described herein are suitable for “topicalapplication” (i.e., on top of skin surface, on top of mucosal surface).As used herein, topical application includes, but is not limited to,cutaneous; ocular; mucosal; buccal; vaginal; vulvar administration;administration onto skin, scar, keloid, scalp, eye, mouth, nose, vulva,vagina, rectum; and/or administration into a wound, ulcer, andgranulation tissue.

The compositions may be suitable for administration to hair, and ontofinger or toe nails. Alternatively, the compositions may be suitable forsubcutaneous administration.

Cosmetic Product

A “cosmetic product,” as used herein, include without limitation,personal care product, skin product, skin cream, skin gel, skinointment, skin lotion, anti-aging product, skin rejuvenation product,skin conditioner, moisturizer, feminine product, hygiene product, skinpatch, skin mask, tissue wipe, lipstick, mascara, rouge, foundation,blush, eyeliner, lip liner, lip gloss, lip balm, facial or body powder,sunscreens, sunblocks, nail polish, mousse, sprays, styling gels, nailconditioner, bath and shower gels, shampoos, conditioners, cream rinses,hair sprays, hair dyes and coloring products, soaps, body scrubs,exfoliants, astringents, depilatories and permanent waving solutions,antidandruff formulations, anti-sweat and antiperspirant compositions,shaving, preshaving and after shaving products, leave-on conditioners,deodorants, cold creams, deodorants, cleansers, rinses, vulvar product,vaginal product, or the like; whether in the form of creams, lotions,gels, ointments, macro-emulsions, micro-emulsions, nano-emulsions,serums, balms, colloids, solutions, liquids, suspensions, dispersions,compacts, solids, powders, pencils, spray-on formulations, brush-onformulations, patches, iontophoretic patches, microprojection patches,microneedle patches, skin delivery enhancing systems, bandage, tissuecloths, wipes, masks, aerosols, pastes, soap bars, cosmetic devices,and/or any other forms readily known to those skilled in the art.

Medicament

A “medicament,” as used herein, include without limitationpharmaceutical preparations, carriers for dermatological purposes,including topical and transdermal application of pharmaceuticalingredients. These can be in the form of creams, lotions, gels,ointments, macro-emulsions, micro-emulsions, nano-emulsions, serums,balms, colloids, solutions, liquids, suspensions, dispersions, compacts,solids, powders, pencils, spray-on formulations, brush-on formulations,patches, iontophoretic patches, microprojection patches, microneedlepatches, skin delivery enhancing systems, bandages, tissue cloths,wipes, masks, aerosols, pastes, soap bars, medical devices, and/or anyother forms readily known to those skilled in the art.

Suitability for Topical Application

The term “acceptable substance(s) for topical application”, as usedherein, mean that the composition(s) comprising “acceptable substance(s)for topical application” according to the invention are suitable for usein contact with human skin and/or human mucosa; where the skin or themucosa can be healthy, newborn, young, old, aged, appear visuallydifferent than normal, damaged, photo-damaged, sunburned, wrinkled,pathologic, diseased, wounded, atrophic, irritated, compromised, treatedwith cosmetic product(s), treated with pharmaceutical product(s),treated with cosmetic procedure(s), treated with dermatologicalprocedure(s), treated with a pharmaceutical or medical device(s),surgically treated, etc. and are absent of significant(consumer-unacceptable) local intolerabilities to skin or mucosa (i.e.,corrosivity, irritation, allergy), and the like after repeated topicalapplication for cosmetic, skin care, feminine, or similar uses; or withmaximally low and acceptable (consumer-acceptable) localintolerabilities to skin or mucosa skin irritation (i.e., irritation,allergy), and the like after repeated topical application for medicaluses of the composition.

Local tolerability (e.g., irritation and allergy to skin; also calledcontact dermatitis and allergy) in humans can be determined by acute (1day) and repetitive (4 to 21 days) patch testing on the back of humans,and/or during in use tests where the composition is used as indicated(e.g., for topical use on face, vulva, vagina, mucosal surface, and/orother body surface areas; or for wound healing). In case of amedication, safety studies generally also include animal studies.

Furthermore, acceptable substance(s) for topical application means thatthe compositions comprising “acceptable substance(s) for topicalapplication” in accordance with the present invention are withoutsignificant physicochemical instability (e.g., significant changes incolor, odor, viscosity, pH, and/or appearance) in the final packaging(e.g., bottle, tube, pump, jar, airless container, spray, patch, etc.)during the shelf-life of the product according to the recommendedstorage conditions of the product. Significant physicochemicalinstability means, that the color, odor, viscosity, pH, or theappearance changed (increased, decreased) more than 10% from the timewhen the composition was prepared and filled into the final packaging.

Any of the compositions of the present invention may also provide goodaesthetics and be cosmetically elegant.

Acceptable substances for topical application or administration mayinclude suitable excipients and/or carriers known in the art.

Additional Substances

The compositions described herein preferably include Octanoyl Carnosine(or one or more derivatives thereof), optionally in combination with atleast one additional substance suitable for topical application and/orsubcutaneous application. Additional substance(s) can be inert (e.g.,carriers and/or d excipients) or can be with biological activities(i.e., biologically active agents and/or active pharmaceuticalingredient). Preferably, the compositions of the invention may alsoinclude additional biological active agents, including, but not limitedto, peptides other than Octanoyl Carnosine, N-Palmitoyl-GHK, and GEKG(SEQ ID NO: 1) (or one or more derivatives thereof) according to thepresent invention.

The terms “substance”, “ingredient”, “agent” and the like are usedinterchangeably herein.

The compositions of the invention may include one or more substances,various, conventional or not, which will provide some benefit to theobject of the composition. More specifically, the combination ofOctanoyl Carnosine (or one or more derivatives thereof) according to thepresent invention with selected additional ingredients may lead to anenhanced efficacy as compared to the use of the Octanoyl Carnosine (orone or more derivatives thereof) alone. The enhanced efficacy can beadditive (the sum of efficacies of the individual agents alone), or itcan be synergistic (larger than the sum of efficacies of the individualagents alone). Of course, the decision to include an additionalingredient and the choice of a specific ingredient depends on thespecific use of the composition and the product formulation and is wellwithin the routine level of skill in the art.

In particular examples, the compositions of the present invention maycontain a wide range of additional ingredients. The 2012 InternationalCosmetic Ingredient Dictionary & Handbook, 14th Edition, as well as theCosmetic Bench Reference—Directory of Cosmetic Ingredients (published byCosmetics & Toiletries) describes a wide variety of non-limitingcosmetic and pharmaceutical ingredients commonly used in the skin care,personal care, feminine care, and dermatology and pharmaceuticalindustry, which are available for use in the present invention.Additional examples can be found in the books provided by the UnitedStates Pharmacopeia (USP), the National Formulary (NF), and otherreferences for cosmetic and pharmaceutical ingredients known in the art.Each of these references is herein incorporated by reference in itsentirety. This information is regularly updated by the addition of newingredients.

Exemplary functional classes of such ingredients are, but are notlimited to, abrasive agent, absorbent powder, absorption base,acidulent, activator, adhesion promoter, agent modulating celldifferentiation, agent modulating cell proliferation, agent stimulatingsynthesis of dermal or epidermal macromolecules, agent preventingdegradation of dermal or epidermal macromolecules, agent acting onmicrocirculation, agent acting on skin barrier, agent acting on energymetabolism of cells, agent increasing the substantivity, antimicrobialsequestering agent, analgesic agent, anesthetic agent, antacid agent,anti-acne agent, anti-aging agent, anti-wrinkle agent, anti-atrophyagent, anti-androgen agent, anti-bacterial agent, anti-scar agent,anti-seborrheic agent, anti-cracking agent, anti-cellulite agent,anti-stretch mark agent anti-dandruff agent, anti-foam agent,anti-fungal agent, anti-histamine agent, anti-inflammatory agent,anti-irritant agent, anti-microbial agent, anti-mite agents, antibioticagent, antiviral agent, antioxidant agent, anti-glycation agent,anti-neoplastic agent, anti-cancer agent, anti-skin cancer agent,anti-eczema agent, anti-psoriasis agent, antipollution agent,antiperspirant agent, anti-pruriginous agent, anti-pruritic agent,antiseptic agent, antistat agent, astringent, α-adrenergic receptoragonist, barrier agent, binding agent, bio-adhesive agents, botanicalagent, botanical extract, biological additive, buffer agent, bulkingagent, calcium sequestering agent, calming agent, carrier agent,chemical additive, cell lysate, cell culture medium, conditioned cellculture medium, chelating agent, circulatory stimulant agent, cleansingagent, collagen stimulating agent, co-emulsifier agent, colorant,conditioning agent, controlled release agent, cooling agent, co-solvent,coupling agent, curative agent, denaturant, deodorant agent, depilatoryagent, desquamating agent, detangler agent, detergent, disinfectant,dispersant, dye stabilizer, dermatologically acceptable carrier, elastinstimulating agent, extracellular matrix stimulating agent, emollient,emulsifier, emulsion stabilizer, enzyme, enzymatic inhibitor,enzyme-inducing agent, coenzyme, cofactor, essential oil, exfoliant, fatsoluble agent, fiber, film former, fixative, flavor, foam booster, foamstabilizer, foaming agent, fragrance, free radicals scavenger,fungicide, gellant, glosser, hair beaching agent, hair growth promoter,hair colorant, hair conditioning agent, hair-set polymer, hormone,hormone-like agent, humectant, hydrophobic agent, hydrotropic agentsintermediate agent, hyaluronic acid stimulating agent, keratolyticagent, lathering agent, lipolytic agent, lubricant, make-up agent,moisture barrier agent, moisturizer, muco-adhesive agents, musclerelaxant, natural moisturizing factor, neutralizer, odor-masking agent,oil, oil absorbent agent, ointment base, opacifier, organosilicone,oxidant, oxygen carrier, pearlant agent, perfume, perfume solvent,perfume stabilizer, peroxide stabilizer, pharmaceutical drug,photo-sensitizer agent, pigment, pigmenting agent, pearlescent aid,plant extract, plant derivative, plant tissue extract, plant rootextract, plant seed extract, plant oil, plasticizer, polish agent,polymer, polymer film former, powder, preservative agent, propellant,peptide agent, protein agent, reducing agent, re-fatting agent,regenerator, resin, rosacea inhibitory agent, scar prevention agent,scalp agent, scrub agent, sabostatic agent, sequestrant, sex hormone,sex stimulating agent, silicone agent, silicone replacement agent, skinbarrier agent, skin barrier restoration agent, skin calming agent, skinclarifier, skin cleanser, skin conditioning agent, skin exfoliatingagent, skin peeling agent, skin healing agent, skin lipid, skinlightening agent, skin bleaching agent, skin protectant agent, skinpurifier agent, skin smoothing agent, skin calming agent, skin soothingagent, skin sensate, skin treatment agent, skin penetration enhancingagent, skin penetration retarding agent, mucosa penetration enhancingagent, solubilizer, solvent, suspending agent, sun protection factorbooster, soothing agent, spreading agent, stabilizer, stimulant agent,slimming agent, sunless tanning agent, sunscreen, sunscreen UVA,sunscreen UVB, broad-band sunscreen, super-fatting agent, surfactant,amphoteric surfactant, anionic surfactant, cationic surfactant,non-ionic surfactant, silicone surfactant, suspending agent, sweetener,tanning accelerator, thickening agent, thixotrope, tightening agent,toner, tonic agent, topical delivery system, vasoconstrictor agent,vulvar soothing agent, vaginal soothing agent, vegetable oil, volatileagent, viscosity stabilizer, vitamin, vaccine, water proofing agent,water-soluble agent, water-proofing agent, wax, wetting agent, whiteningagent, wound healing agent, and/or the like.

Preferably, the additional ingredients should be suitable for use incontact with human keratinous tissue (hair, nails, skin, lips, externalvulva (mons pubis, labia majora, labia minora)) and/or non-keratinoustissue (vagina, introitus, inner vulva (vulvar vestibule, clitoris),mouth, anus, etc.), without undue systemic toxicity localintolerability, and chemical instability.

In most instances, the additional substances will include a cosmetic,dermatologically, and/or pharmaceutically acceptable carrier eitheralone or in combination with still other additional (e.g., inert and/orbiologically active) ingredients. The total amounts of additionalingredients may range from about 90% to about 99.9999%, preferably fromabout 95% to about 99.999%, and more preferably from about 99% to about99.999%, of the composition. In short, it is the balance of thecomposition. If carriers (either singularly, such as water, or complexco-solvents) are used, they may make up the entire balance of thecompositions.

Non-limiting examples of additional ingredients for some of thefunctional classes listed above are provided herein. Additional examplesof additional ingredients can be found in The International CosmeticIngredient Dictionary and Handbook, the Cosmetic BenchReference—Directory of Cosmetic Ingredients, the books provided by theUnited States Pharmacopeia (USP) and the National Formulary (NF), andother references for cosmetic and pharmaceutical ingredients known (andcommonly used) in the art.

In order to be suitable for use in accordance with the presentinvention, the additional ingredients and carrier/excipients must befurther chemically compatible with Octanoyl Carnosine (or one or morederivatives thereof), with Palmitoyl-GHK (or one or more derivativesthereof), and/or with GEKG (SEQ ID NO: 1) (or one or more derivativesthereof). Here, “chemically compatible” means that the additionalingredients do not lead to a significant chemical degradation (e.g.,hydrolysis, oxidation). For example, a significant chemical degradationwould include more than 10% degradation during the shelf-life period(e.g., as provided by the expiration date) of the product under therecommended storage conditions of the product.

Peptides

The composition of the present invention can contain additionalpeptide(s). Suitable peptides can include, but are not limited to, di-,tri-, tetra-, penta-, hexa-peptides, and other oligo- to poly-peptides,and derivatives thereof.

For example, when included in the present compositions, the additionalpeptides are preferably used in amounts ranging from about 0.000001% toabout 10%, more preferably from about 0.000001% to about 1%, and evenmore preferably from about 0.00001% to about 0.1% by weight of thecomposition. The exact content (%) of peptides to be used in thecompositions will depend on the particular peptide utilized since suchagents vary widely in potency.

Suitable dipeptides for use herein include, but are not limited to,carnosine (beta-Ala-His), Tyr-Arg, Val-Trp (see WO 0164178), Asn-Phe,Asp-Phe. Suitable tripeptides for use herein include, but are notlimited to, Arg-Lys-Arg, His-Gly-Gly, Gly-His-Lys, Gly-Gly-His,Gly-His-Gly, Lys-Phe-Lys. Suitable tetrapeptides for use herein include,but are not limited to, Peptide E, Arg-Ser-Arg-Lys (SEQ ID NO: 10),Gly-Gln-Pro-Arg (SEQ ID NO:9). Suitable pentapeptides include, but arenot limited to, Lys-Thr-Thr-Lys-Ser (SEQ ID NO: 11). Suitablehexapeptides include, but are not limited to, Val-Gly-Val-Ala-Pro-Gly(SEQ ID NO: 12) and such as those disclosed in FR 2854897 and US2004/0120918.

Other suitable peptides for use herein include, but are not limited to,lipophilic derivatives of peptides, preferably octanoyl, decanoyl,lauroyl, myristoyl and palmitoyl derivatives, and metal complexes of theaforementioned (e.g., copper complex of the tripeptide His-Gly-Gly).Preferred dipeptide derivatives include N-Palmitoyl-beta-Ala-His,N-Acetyl-beta-Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (WO 9807744, U.S.Pat. No. 6,372,717). Preferred tripeptide derivatives include the copperderivative of His-Gly-Gly, N-Elaidoyl-Lys-Phe-Lys and its analogs ofconservative substitution, N-Acetyl-Arg-Lys-Arg-NH2, N-Biot-Gly-His-Lys(WO0058347) and derivatives thereof. Suitable tetrapeptide derivativesfor use herein include, but are not limited toN-palmitoyl-Gly-Gln-Pro-Arg (SEQ ID NO:9), suitable pentapeptidederivatives for use herein include, but are not limited toN-Palmitoyl-Lys-Thr-Thr-Lys-Ser (SEQ ID NO:11); WO 0015188 and U.S. Pat.No. 6,620,419), N-Palmitoyl-Tyr-Gly-Gly-Phe-X with X Met or Leu (SEQ IDNO: 13), or mixtures thereof. Suitable hexapeptide derivatives for useherein include, but are not limited toN-Palmitoyl-Val-Gly-Val-Ala-Pro-Gly (SEQ ID NO:12) and derivativesthereof.

Preferred dipeptide derivatives include N-Palmitoyl-beta-Ala-His,N-Acetyl-beta-Ala-His, N-Acetyl-Tyr-Arg-hexadecylester (WO 9807744, U.S.Pat. No. 6,372,717). Preferred tripeptide derivatives include the copperderivative of His-Gly-Gly, N-Elaidoyl-Lys-Phe-Lys and its analogs ofconservative substitution, N-Acetyl-Arg-Lys-Arg-NH2, N-Biot-Gly-His-Lys(WO0058347) and derivatives thereof. Suitable tetrapeptide derivativesfor use herein include, but are not limited toN-palmitoyl-Gly-Gln-Pro-Arg, suitable pentapeptide derivatives for useherein include, but are not limited to N-Palmitoyl-Lys-Thr-Thr-Lys-Ser(WO 0015188 and U.S. Pat. No. 6,620,419), N-Palmitoyl-Tyr-Gly-Gly-Phe-Xwith X Met or Leu (SEQ ID NO: 13), or mixtures thereof. Suitablehexapeptide derivatives for use herein include, but are not limited toN-Palmitoyl-Val-Gly-Val-Ala-Pro-Gly and derivatives thereof.

The peptides can be obtained from any supplier of commercially availablecosmetic and pharmaceutical peptides, peptide mixtures or derivativesthereof; including but not limited to Atrium, Unipex, Lucas MeyerCosmetics, Biotechnologies, Sederma, Croda, Grant Industries,Pentapharm, DSM, Evonik, Lipotec, Symrise, BASF, ISP, Helix BioMedix,Oriflame, Seppic, Solabia, Procyte, EMD Chemicals, Corium Peptides,etc.; or can be directly obtained by custom synthesis. When usingcommercially available cosmetic and pharmaceutical peptides, thepreferred composition generally contains the additional peptide(s) inthe concentration range as recommended by the peptide supplier.

Additional examples of suitable peptides can be also found in thechapter by F. Gorohhui and H. I. Maibach in the Textbook of Aging (2010,Springer), in Clinics in Dermatology 2009, 27, 485-495, or numerousother scientific articles, communications, patent applications, grantedpatents on peptides for cosmetic or medical uses (incorporated herein byreference).

Ascorbates and Other Vitamins

The compositions of the present invention may contain one or morevitamins, such as ascorbates (e.g., vitamin C, vitamin C derivatives,ascorbic acid, ascorbyl glucoside, ascorbyl palmitate, magnesiumascorbyl phosphate, sodium ascorbyl phosphate, tetrahexadecyl ascorbate,ascorbyl 3-aminopropyl phosphate), vitamin B, vitamin B derivatives,vitamin B1 to vitamin B12 and theirs derivatives, vitamin K, vitamin Kderivatives, vitamin H, vitamin D, vitamin D3, vitamin D derivatives,vitamin E, vitamin E derivatives, and pro-vitamins thereof, such aspanthenol and mixtures thereof. The vitamin compounds may be included asthe substantially pure material, or as an extract obtained by suitablephysical and/or chemical isolation from natural (e.g., plant) sources.In one example, when vitamin compounds are present in the compositionsof the instant invention, the compositions contain from about 0.0001% toabout 25%, more preferably from about 0.001% to about 10%, still morepreferably from about 0.01% to about 5%, and still more preferably fromabout 0.1% to about 1%, by weight of the composition, of the vitamincompound. The exact content (%) of ascorbates and other vitamins to beused in the compositions will depend on the particular ascorbate andvitamin utilized since such agents vary widely in potency.

Sunscreen Actives

The compositions of the subject invention may optionally contain asunscreen active. As used herein, “sunscreen active” includes bothsunscreen agents and physical sunblocks. Suitable sunscreen actives maybe organic or inorganic. A wide variety of conventional organic orinorganic sunscreen actives are suitable for use herein. In one example,the composition contains from about 0.1% to about 25%, more typicallyfrom about 0.5% to about 10% by weight of the composition, of thesunscreen active. Exact amounts will vary depending upon the sunscreenchosen and the desired Sun Protection Factor (SPF). The organicUV-screening agents which are more particularly preferred are chosenfrom the following compounds: ethylhexyl salicylate, butylmethoxydibenzoylmethane, ethylhexyl methoxycinnamate, octocrylene,phenylbenzimidazole sulphonic acid, terephthalylidene dicamphorsulphonic, benzophenone-3, benzophenone-4,benzophenone-5,4-methylbenzylidene camphor, benzimidazilate,anisotriazine, Ethylhexyl triazone, diethylhexyl butamido triazone,methylene bis-benzotriazolyl tetramethylbutylphenol, drometrizoletrisiloxane, and mixtures thereof.

The inorganic sunscreen agents which may be used in the compositionaccording to the invention are in particular nanopigments (mean size ofthe primary particles: generally between 5 nm and 100 nm, preferablybetween 10 nm and 50 nm; or their aggregates) of coated or uncoatedmetal oxides such as for example nanopigments of titanium oxide(amorphous or crystallized in the form of rutile and/or anatase), iron,zinc, zirconium or cerium oxides and mixtures thereof. Coating agentsare moreover alumina and/or aluminum stearate, and silicones.

Anti-Wrinkle Actives and Anti-Atrophy Actives

The compositions of the present invention can contain a one or moreanti-wrinkle actives or anti-atrophy actives. Exemplaryanti-wrinkle/anti-atrophy actives suitable for use in the compositionsof the present invention include amino acids, N-acetyl derivatives ofamino acids (e.g., N-acetyl-cysteine), hydroxy acids (e.g.,alpha-hydroxy acids such as lactic acid and glycolic acid orbeta-hydroxy acids such as salicylic acid and salicylic acid derivativessuch as the octanoyl derivative, lactobionic acid), keto acids (e.g.,pyruvic acid), phytic acid, ascorbic acid (vitamin C), retinoids (e.g.,retinoic acid, tretinoin, isotretinoin, adapalene, retinol,retinylaldehyde, retinylpalmitate, and other retinoid derivatives),kinetin (N6-furfuryladenine), zeatin and their derivatives (e.g.,furfurylamino-tetrahydropyranyladenine), niacinamide (nicotinamide);growth factors and cytokines (e.g., TGF-beta 1, 2 and 3, EGF, FGF-2,PDGF, IL-1, IL-6, IL-8, IGF-1, IGF-2, etc.), cell lysates (e.g., dermalfibroblast cell lysate, stem cell lysate, processed skin cell proteins(PSP®), etc.), conditioned cell culture mediums (e.g., conditioned cellculture medium from dermal fibroblasts, conditioned cell culture mediumfrom stem cells (e.g., epidermal stem cells, adipose stem cells,mesenchymal stem cells, etc.); cosmetic ingredients marketed under thetrade names Nouricel-MD®, TNS®, or CCM™ Complex; etc.); cell extracts,stem cell extracts, components from stem cells; ingredients stimulatingepidermal or other human adult stem cells; skin conditioning agents,stilbenes, cinnamates, ingredients activating sirtuin 1 (e.g.,resveratrol); ingredients improving the functioning of the mitochondria;dimethylaminoethanol, synthetic anti-aging peptides, peptides fromnatural sources (e.g., soy peptides), and salts of sugar acids (e.g., Mngluconate, Zn gluconate), lipoic acid; lysophosphatidic acid, vitamin B3compounds, and other vitamin B compounds (e.g., thiamine (vitamin B1),pantothenic acid (vitamin B5), riboflavin (vitamin B2), and theirderivatives and salts (e.g., HCl salts or calcium salts).

When anti-wrinkle/anti-atrophy compounds are present in the compositionsof the instant invention, the compositions comprise from about 0.0001%to about 25%, more preferably from about 0.001% to about 10%, still morepreferably from about 0.01% to about 5%, and still more preferably fromabout 0.1% to about 1%, by weight of the composition, of theanti-wrinkle/anti-atrophy compound. The exact content (%) ofanti-wrinkle/anti-atrophy agents to be used in the compositions willdepend on the particular anti-wrinkle/anti-atrophy agent utilized sincesuch agents vary widely in potency.

Humectants, Moisturizers, and Conditioning Agents

The compositions of the present invention can contain a safe andeffective amount of a conditioning agent selected from, for example,humectants, moisturizers, and skin conditioners. A variety of thesematerials can be employed and can be present at a level of from about0.01% to about 80%, more preferably from about 0.1% to about 25%, andstill more preferably from about 0.5% to about 10%, by weight of thecomposition. The exact content (%) of humectants, moisturizers, andconditioning agents to be used in the compositions will depend on thehumectant, moisturizer, and conditioning agent utilized since suchagents vary widely in potency.

Humectants are ingredients that help maintain moisture levels in skin.Humectants can be selected from the group consisting of polyhydricalcohols, water soluble alkoxylated nonionic polymers, and mixturesthereof. Polyhydric alcohols useful herein include polyhdroxy alcoholsaforementioned and glycerin, hexylene glycol, ethoxylated glucose,1,2-hexane diol, dipropylene glycol, trehalose, diglycerin, maltitol,maltose, glucose, fructose, sodium chondroitin sulfate, sodiumhyaluronate, sodium adenosine phosphate, sodium lactate, pyrrolidonecarbonate, glucosamine, cyclodextrin, and mixtures thereof. Watersoluble alkoxylated nonionic polymers useful herein include polyethyleneglycols and polypropylene glycols having a molecular weight of up toabout 1000 such as those with CTFA names PEG-200, PEG-400, PEG-600,PEG-1000, and mixtures thereof. Additional humectants include acetylarginine, algae extract, aloe barbadensis leaf extract, 2,3-butanediol,chitosan lauroyl glycinate, diglycereth-7 malate, diglycerin, diglycolguanidine succinate, erythritol, fructose, glucose, glycerin, honey,hydrolyzed proteins, hydroxypropyltrimonium hyaluronate, inositol,lactitol, maltitol, maltose, mannitol, mannose, methoxy polyethyleneglycol, myristamidobutyl guanidine acetate, polyglyceryl sorbitol,potassium pyrollidone carboxylic acid (PCA), propylene glycol, butyleneglycol, sodium pyrollidone carboxylic acid (PCA), sorbitol, sucrose,dextran sulfate (i.e., of any molecular weight), natural moisturizingfactors, and/or urea.

Skin conditioners can include, but are not limited to, guanidine, urea,glycolic acid, glycolate salts (e.g., ammonium and quaternary alkylammonium), salicylic acid, lactic acid, lactate salts (e.g., ammoniumand quaternary alkyl ammonium), aloe vera in any of its variety of forms(e.g., aloe vera gel), polyhydroxy alcohols such as sorbitol, mannitol,xylitol, erythritol, hexanetriol, butanetriol, propylene glycol,butylene glycol, hexylene glycol and the like, polyethylene glycols,propoxylated glycerols, sugars (e.g., melibiose), starches, sugar andstarch derivatives (e.g., alkoxylated glucose, fructose, glucosamine),C1-C30 monoesters and polyesters of sugars and related materials,hyaluronic acid, lactamide monoethanolamine, acetamide monoethanolamine,panthenol, dexpanthenol, allantoin, and mixtures thereof. Skinconditioners can also include fatty acids, fatty acid esters, lipids,ceramides, cholesterol, cholesterol esters, bee wax, petrolatum, andmineral oil.

Emollients

One or more emollients may also be included in the topical compositionsdescribed herein. An emollient generally refers to an ingredient thatcan help skin maintain a soft, smooth, and pliable appearance.Emollients typically remain on the skin surface, or in the stratumcorneum, and act as a moisturizer, or lubricant and reduce flaking. Someexamples of emollients include acetyl arginine, acetylated lanolin,algae extract, apricot kernel oil polyethylene glycol-6 esters, avocadooil polyethylene glycol-11 esters, bis-polyethylene glycol-4dimethicone, butoxyethyl stearate, glycol esters, alkyl lactates,caprylyl glycol, cetyl esters, cetyl laurate, coconut oil polyethyleneglycol-10 esters, alkyl tartrates, diethyl sebacate, dihydrocholesterylbutyrate, dimethiconol, dimyristyl tartrate, disteareth-5 lauroylglutamate, ethyl avocadate, ethylhexyl myristate, glyceryl isostearates,glyceryl oleate, hexyldecyl stearate, hexyl isostearate, hydrogenatedpalm glycerides, hydrogenated soy glycerides, hydrogenated tallowglycerides, isostearyl neopentanoate, isostearyl palmitate, isotridecylisononanoate, laureth-2 acetate, lauryl polyglyceryl-6 cetearyl glycolether, methyl gluceth-20 benzoate, mineral oil, palm oil, coconut oil,myreth-3 palmitate, octyldecanol, octyldodecanol, odontella aurita oil,2-oleamido-1,3 octadecanediol, palm glycerides, polyethylene glycolavocado glycerides, polyethylene glycol castor oil, polyethyleneglycol-22/dodecyl glycol copolymer, polyethylene glycol shea butterglycerides, phytol, raffinose, stearyl citrate, sunflower seed oilglycerides, petrolatum, silicon oils including but not limited tocaprylyl methicone, and/or tocopheryl glucoside.

Anti-Oxidants, and Radical Scavengers

The compositions of the present invention may include ananti-oxidant/radical scavenger. In one example, the composition containsfrom about 0.001% to about 25%, more preferably from about 0.01% toabout 10%, and still more preferably from about 0.1% to about 5%, of ananti-oxidant/radical scavenger. The exact content (%) ofanti-oxidant/radical scavengers to be used in the compositions willdepend on the particular anti-oxidant/radical scavenger utilized sincesuch agents vary widely in potency.

Anti-oxidants/radical scavengers may include but are not limited toascorbic acid (vitamin C) and its salts, ascorbyl esters of fatty acids,and other ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate,sodium ascorbyl phosphate, ascorbyl sorbate, ascorbyl palmitate,tetrahexyldecyl ascorbate, etc.), tocopherol (vitamin E), tocopherolsorbate, tocopherol acetate, other esters of tocopherol, beta-carotene,butylated hydroxy benzoic acids and their salts, ferulic acid, peroxidesincluding hydrogen peroxide, perborate, thioglycolates, persulfatesalts, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid(commercially available under the trade name Trolox™), gallic acid andits alkyl esters, especially propyl gallate, uric acid and its salts andalkyl esters, amines (e.g., N,N-diethylhydroxylamine, amino-guanidine),nordihydroguaiaretic acid, bioflavonoids, sulfhydryl compounds (e.g.,glutathione), dihydroxy fumaric acid and its salts, lysine pidolate,arginine pilolate, amino acids, silymarin, lysine, 1-methionine,proline, superoxide dismutase, sorbic acids and its salts, lipoic acid,olive extracts, tea extracts, resveratrol, polyphenols such asproanthocyanidine from pine bark, carotenoids, curcumin compounds suchas tetrahydrocurcumin, coenzyme Q10, OCTA (L-2-oxo-4-thiazolidinecarboxylic acid), selenium, creatine, glutathione, N-acetyl cysteine,N-acetyl cysteine esters, dimethylmethoxy chromanol, lipoic acid,melanin; plant extracts containing polyphenols including but not limitedto coffee berry extracts, green tea extracts, rosemary extracts, witchhazel extracts, and grape skin/seed extracts, may be used. Preferredanti-oxidants/radical scavengers can be selected from esters of ascorbicacid, tocopherol, ferulic acid, polyphenols, creatine, and theirderivatives; as well as plant extracts containing polyphenols such asgreen tea extract.

Antimicrobial Peptide Sequestering Agents

Antimicrobial peptide sequestering compounds may include but are notlimited to a sulfated or polysulfated monosaccharide, and salts andcomplexes thereof; a sulfated or polysulfated disaccharide, and saltsand complexes thereof; a sulfated or polysulfated polysaccharide, andsalts and complexes thereof; a dextran sulfate (e.g., sodium dextransulfate), and salts and complexes thereof; chondroitin sulfate, andsalts and complexes thereof; pentosan polysulfate, and salts andcomplexes thereof; sucrose sulfate (e.g., any sucrose sulfate such assucrose octasulphate other than aluminum sucrose sulfate), and salts andcomplexes thereof; a fucoidan, and salts and complexes thereof; asulfated galactan, and salts and complexes thereof; a carrageenans(e.g., Chondrus Crispus), and salts and complexes thereof; starchsulfate, and salts and complexes thereof; cellulose sulfate, and saltsand complexes thereof; a sulfated glycosaminoglycan, and salts andcomplexes thereof; a heparin; a heparan sulfate; sulfated glucan; or anycombinations thereof. The antimicrobial peptide sequestering compoundmay include a plant extract, an algae extract, an aloe vera(barbadensis) extract, a cactus extract, or a shark or fish cartilageextract. The antimicrobial peptide sequestering compound may also be asulfated or polysulfated polymer (e.g., poly(vinyl sulfate),poly(anethole sulfonate)). Suitable polymeric sulfonic acid that can beused in the methods and compositions described herein arehydrophobically modified polymeric sulfonic acids such as Aristoflex®HMP or Aristoflex® AVC (Clariant). Alternatively, the antimicrobialpeptide sequestering compound is a phosphate or polyphosphate (e.g., amonosaccharide phosphate, a disaccharide phosphate, a polysaccharidephosphate, a glycerophosphate salt, or a starch phosphate). Suitableexamples of starch phosphates include, but are not limited tohydroxypropyl starch phosphates (i.e., Structure XL (National Starch,LCC)). The antimicrobial peptide sequestering compound may also be aphospholipid such as phosphatidylcholine or lecithin. Further, theantimicrobial peptide sequestering compound can be a carboxylate, apolyhydroxy acid, hyaluronic acid, alginate, and/or polylactic acid.Most preferably, the antimicrobial peptide sequestering compounds arebetween 100 to 10,000 g per mol. Sodium dextran sulfate of about 5000 to10′000 g per mol is one of the most preferred antimicrobial peptidesequestering compound.

Rosacea Inhibitory Agents, and α-Adrenergic Receptor Agonists

Rosacea inhibitory agents, include but are not limited to,metronidazole, sulfacetamide, sodium sulfacetamide, sulfur, dapson,doxycycline, minocycline, clindamycin, clindamycin phosphate,erythromycin, tetracylclines, azelaic acid, calcium dobesilate, maleicacid, and any compatible combinations thereof); α-adrenergic receptoragonists (e.g., clonidine, amphetamine, doxtroamphetamine,apraclonidine, dipivefrin, α-methyldopa, oxymetazoline, oxymetazolinehydrochloride, methoxamine, metaraminol, medetomidine, dexmedetomidine,ethylnorepinephrine, guanfacine, guanabenz, phenylephrine, phenylephrinehydrochloride, ephedrine, epinine, epinephrine, ethylnorepinephrine,levarterenol, lofexidine, norepinephrine, norphenylephrine,norephedrine, phenylpropanolamine, pemoline, propylhexadrine,pseudoephedrine, methamphetamine, α-methylnorepinephrine,methylphenidate, mephentermine, midodrine, mivazerol, moxonidine,desglymidodrine, tetrahydrozoline, tetrahydrozoline hydrochloride,cirazoline, amidephrine, brimonidine, brimonidine tartrate, naphazoline,isoproterenol, xylazine, xylometazoline, and/or tizanidine); chemicalsand botanical extracts with vasoconstrictor properties including, butnot limited to, corticosteroids, ephedrine, pseudoephedrine, caffeine,and/or escin; ephedra, phedra sinica, hamamelis viginiana, hydrastiscanadensis, lycopus virginicus, aspidosperma quebracho, cytisusscoparius, raphanus sativus linn (radish leave extracts), horse chestnutextracts, etc., as well as any compatible combinations thereof; and/or anasal and/or sinus decongestant.

Skin Lightening Agents, and Skin Bleaching Agents

The compositions of the present invention may contain a skin lighteningagent. Suitable skin lightening agents include, but are not limited to,ascorbic acid and derivatives thereof; kojic acid and derivativesthereof; resorcinol and derivatives thereof (including but not limitedto 4-ethyl resorcinol, 4-butyl resorcinol, 4-hexyl resorcinol, 4-octylresorcinol, 4-decyl resorcinol, 6-methyl resorcinol, 6-ethyl resorcinol,6-butyl resorcinol, 6-hexyl resorcinol, 6-octyl resorcinol, 6-decylresorcinol, 4-phenylethyl resorcinol), retinoic acid and derivativesthereof (e.g., retinol, retinyl palmitate), L-leucine and derivativesthereof (e.g., N-acyl derivatives of L-leucine, esters of L-leucine,etc.), glycine and derivatives thereof, disodium glycerophosphate andderivatives thereof, undecenoyl phenylalanine, arbutin and derivativesthereof (e.g., dehydroxyarbutin), niacinamide and derivatives thereof,hydroquinone; mequinol, glabridin, aleosin, curcumin, genistein, ethyllinoleate, tranexaminic acid, azelaic acid, resveratrol and derivativesthereof (e.g., oxyresveratrol), N-acetyl glucosamine,4-isopropylcetchol, 4-ethoxybenzaldehyde, 2-ethoxybenzaldehyde,4-propoxybenzaldehyde, alpha-hydroxyacids (e.g., glycolic acid, lacticacid, etc.), salicylic acid, polyphenols; and/or various plant extracts,such as those from licorice, grape seed, mulberry, soy, green tea,and/or bear berry; and/or any ingredient or combination thereof.

When used, the compositions preferably contain from about 0.01% to about15%, more preferably from about 0.1% to about 10%, also preferably fromabout 0.5% to about 5%, by weight of the composition, of a skinlightening agent. The exact content (%) of skin lightening agents to beused in the compositions will depend on the particular skin lighteningagent utilized since such agents vary widely in potency.

Skin Protectants

Suitable skin protectant agents for use in the compositions describedherein include, for example, a compound that protects injured or exposedskin or mucous membrane surfaces from harmful or irritating externalcompounds. Representative examples include algae extract, allantoin,Camellia sinensis leaf extract, cerebrosides, dimethicone,glucuronolactone, glycerin, kaolin, lanolin, malt extract, mineral oil,petrolatum, white petrolatum, potassium gluconate, colloidal oat meal,calamine, cocoa butter, starch, zinc oxide, zinc carbonate, zincacetate, and/or talc.

Desquamation Actives, Keratolytic Agents, and Peeling Agents

A desquamating/keratolytic active may be added to the compositions ofthe present invention. In one example, the composition contains fromabout 0.01% to about 30%, preferably from about 0.1% to about 10%, morepreferably from about 0.5% to about 5%, by weight of the composition, ofa desquamating/keratolytic active. The exact content (%) ofdesquamating/keratolytic agents to be used in the compositions willdepend on the particular desquamating/keratolytic agent utilized sincesuch agents vary widely in potency.

Examples of useful keratolytic and/or desquamating agents include urea,salicylic acid and alkyl derivatives thereof, saturated and unsaturatedmonocarboxylic acids, saturated and unsaturated bicarboxylic acids,tricarboxylic acids, alpha hydroxyacids and beta hydroxyacids ofmonocarboxylic acids, alpha hydroxyacids and beta hydroxyacids ofbicarboxylic acids, alpha hydroxyacids and beta hydroxyacids oftricarboxylic acids, ketoacids, alpha ketoacids, beta ketoacids, of thepolycarboxylic acids, of the polyhydroxy monocarboxylic acids, of thepolyhydroxy bicarboxylic acids, of the polyhydroxy tricarboxylic acids.Resorcinol and its low-molecular weight derivatives are other examplesof useful keratolytic and/or desquamating agents.

Preferred keratolytic agents are selected from the group containingglycolic acid, tartaric acid, salicylic acid, citric acid, lactic acid,pyruvic acid, gluconic acid, glucuronic acid, malic acid, mandelic acid,oxalic acid, malonic acid, succinic acid, acetic acid, phenol,resorcinol, retinoic acid, adapalene, trichloroacetic acid, 5-fluorouracil, azelaic acid. Keratolytic agents are also the salts, esters,possible cis- or trans-forms, racemic mixtures and/or the relativedextrorotatory or levorotatory forms of the above listed compounds. Suchsubstances can be used singularly or in associations with each other.

Anti-Inflammatory Agents

An anti-inflammatory agent may be added to the compositions of thepresent invention. In one example, an anti-inflammatory agent is addedat a level of from about 0.01% to about 10%, preferably from about 0.5%to about 5%, by weight of the composition. The exact content (%) ofanti-inflammatory agents to be used in the compositions will depend onthe particular anti-inflammatory agent utilized since such agents varywidely in potency.

Steroidal anti-inflammatory agents can include, but are not limited to,corticosteroids such as hydrocortisone, hydroxyltriamcinolone,alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasonedipropionates, clobetasol valerate, desonide, desoxymethasone,desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasonediacetate, diflucortolone valerate, fluadrenolone, flucloroloneacetonide, fludrocortisone, flumethasone pivalate, fluosinoloneacetonide, fluocinonide, flucortine butylesters, fluocortolone,fluprednidene (fluprednylidene) acetate, fluradrenolone, halcinonide,hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone,triamcinolone acetonide, cortisone, cortodoxone, flucetonide,fludrocortisone, difluorosone diacetate, fluradrenolone,fludrocortisone, diflurosone diacetate, fluradrenolone acetonide,medrysone, amcinafel, amcinafide, betamethasone and the balance of itsesters, chloroprednisone, chlorprednisone acetate, clocortelone,clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide,fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate,triamcinolone, and mixtures thereof may be used. One of the preferredsteroidal anti-inflammatory for use is hydrocortisone.

In addition, non-steroidal anti-inflammatory agents can be usefulherein. The varieties of compounds encompassed by this group are wellknown to those skilled in the art. Specific non-steroidalanti-inflammatory agents that can be useful in the composition of thepresent invention include, but are not limited to, diclofenac,indomethacin, oxicams such as piroxicam, salicylates such as aspirin;acetic acid derivatives such as felbinac, fenamates such as etofenamate,flufenamic acid, mefenamic acid, meclofenamic acid, tolfenamic acid;propionic acid derivatives such as ibuprofen, naproxen, pyrazoles, andmixtures thereof. Mixtures of these non-steroidal anti-inflammatoryagents may also be employed, as well as the dermatologically acceptablesalts and esters of these agents. For detailed disclosure of thechemical structure, synthesis, side effects, etc. of non-steroidalanti-inflammatory agents, one may refer to standard texts, includingAnti-inflammatory and Anti-Rheumatic Drugs, K. D. Rainsford, Vol. I-III,CRC Press, Boca Raton, (1985), and Anti-inflammatory Agents, Chemistryand Pharmacology, 1, R. A. Scherrer, et al., Academic Press, New York(1974).

Finally, so-called “natural” anti-inflammatory agents are useful inmethods of the present invention. Such agents may suitably be obtainedas an extract by suitable physical and/or chemical isolation fromnatural sources (e.g., plants, fungi, by-products of microorganisms) orcan be synthetically prepared. For example, candelilla wax, bisabolol(e.g., alpha bisabolol), aloe vera, plant sterols (e.g., phytosterol),kola extract, chamomile, red clover extract, sea whip extract, licoriceextract, and tea extract may be used.

Anti-inflammatory agents useful herein include allantoin and compoundsof the Licorice, including glycyrrhetic acid, glycyrrhizic acid, andderivatives thereof (e.g., salts and suitable esters). Additionalanti-inflammatory agents include diosgenol, saponines, sapogenines,lignanes, triterpenes saponosides and genines.

Additional examples of anti-inflammatory agents can includeanti-inflammatory interleukins (e.g., IL-ira, IL-10); anti-inflammatoryfatty acids (e.g., linoleic acid, linolenic acid) and their derivatives(e.g., esters), isoprenylcystein analogues (i.e.,N-acetyl-S-farnesyl-L-cysteine), aromatic aldehydes withanti-inflammatory properties (e.g., 4-methoxy benzaldehyde, 4-ethoxybenzaldehyde, 4-butoxy benzaldehyde, 4-penthoxy benzaldehyde), as wellas any compatible combinations thereof.

Anti-Acne Actives

The compositions of the present invention can contain one or moreanti-acne actives. Examples of useful anti-acne actives includeresorcinol, sulfur, erythromycin, salicylic acid, benzoyl peroxide,retinoic acid, tretinoin, alpha-hydroxy acids (e.g., glycolic acid,lactic acid), dehydroacetic acid and zinc. When anti-acne compounds arepresent in the compositions of the instant invention, the compositionscontain from about 0.0001% to about 50%, more preferably from about0.001% to about 20%, still more preferably from about 0.01% to about10%, and still more preferably from about 0.1% to about 5%, by weight ofthe composition, of the anti-acne compound. The exact content (%) ofanti-acne actives to be used in the compositions will depend on theparticular antimicrobial, anti-bacterial and anti-acne active utilizedsince such agents vary widely in potency.

Antimicrobial, Anti-Bacterial and Anti-Fungal Actives

The compositions of the present invention can contain one or moreanti-fungal or anti-microbial actives. A safe and effective amount of anantimicrobial or antifungal active can be added to the presentcompositions. For example, the composition contains from about 0.001% toabout 10%, preferably from about 0.01% to about 5%, and more preferablyfrom about 0.05% to about 2%, by weight of the composition, of anantimicrobial or antifungal active. The exact content (%) ofantimicrobial, anti-bacterial and anti-fungal actives to be used in thecompositions will depend on the particular antimicrobial, anti-bacterialand anti-fungal active utilized since such agents vary widely inpotency.

Suitable anti-microbial actives include, but are not limited coal totar, sulfur, aluminum chloride, gentian violet, octopirox (piroctoneolamine), 3,4,4′-trichlorocarbanilide (trichlosan), triclocarban,ciclopirox olamine, undecylenic acid and it's metal salts, potassiumpermanganate, selenium sulphide, sodium thiosulfate, propylene glycol,oil of bitter orange, urea preparations, griseofulvin,8-hydroxyquinoline ciloquinol, thiobendazole, thiocarbamates,haloprogin, polyenes, hydroxypyridone, morpholine, benzylamine,allylamines (such as terbinafine), tea tree oil, clove leaf oil,coriander, palmarosa, berberine, thyme red, cinnamon oil, cinnamicaldehyde, citronellic acid, hinokitol, ichthyol pale, iodopropynylbutylcarbamate, azelaic acid, isothiazalinones such as octylisothiazolinone and azoles, parabens (e.g., methylparaben, ethylparaben,etc.), glycols (e.g., hexylenglycol, ethylhexylglycerin), andcombinations thereof.

For example, suitable agents with anti-fungal properties areketoconazole, naftifine hydrochloride, oxiconazole nitrate, sulconazolenitrate, urea, terbinafine hydrochloride, selenium sulfide. Suitableagents with anti-mite properties are crotamiton, ivermectin, andpermethrin.

One or more anti-fungal or anti-microbial active is combined with ananti-dandruff active selected from polyvalent metal salts of pyrithione.

Anesthetics

The compositions of the present invention may also contain a safe andeffective amount of a topical anesthetic. Examples of topical anestheticdrugs include benzocaine, lidocaine, bupivacaine, chlorprocaine,dibucaine, etidocaine, mepivacaine, tetracaine, dyclonine, hexylcaine,procaine, cocaine, ketamine, pramoxine, phenol; and pharmaceuticallyacceptable salts thereof; benzyl alcohol, camphor, menthol, resorcinol;and appropriate combinations thereof.

Plant Extracts and Vegetable Extracts

The compositions of the present invention may also contain a safe amountof a plant extract and vegetable extract. Examples of plant or vegetableextracts include extracts obtained from ivy (in particular English Ivy(Hedera Helix)), Chinese thorowax (Bupleurum chinensis), barley,Bupleurum Falcatum, arnica (Arnica Montana L), rosemary (Rosmarinusofficinalis N), marigold (Calendula officinalis), sage (Salviaofficinalis L), soy, ginseng (Panax ginseng), ginko biloba,St.-John's-Wort (Hyperycum Perforatum), butcher's-broom (Ruscusaculeatus L), European meadowsweet (Filipendula ulmaria L), big-floweredJarva tea (Orthosiphon Stamincus Benth), algae (Fucus Vesiculosus),birch (Betula alba), green tea, white tea, fermented tea, cola nuts(Cola Nipida), horse-chestnut, bamboo, spadeleaf (Centella asiatica),heather, fucus, willow, witch hazel, wild yam, mouse-ear, escine,cangzhu, chrysanthellum indicum, plants of the Armeniacea genus,Atractylodis Platicodon, Sinnomenum, Pharbitidis, Flemingia, Coleus suchas C. Forskohlii, C. blumei, C. esquirolii, C. scutellaroides, C.xanthantus and C. Barbatus, root of Coleus barbatus, Ballote, Guioa,Davallia, Terminalia, Barringtonia, Trema, antirobia, cecropia, argania,dioscoreae such as Dioscorea opposita or Mexican, Ammi visnaga, Centellaasiatica and Siegesbeckia, in particular Siegesbeckia orientalis, thefamily of Ericaceae in particular bilberry extracts (Vacciniumangustifollium) or Arctostaphylos uva ursi, aloe vera, plant sterols(e.g., phytosterol), Manjistha (extracted from plants in the genusRubia, particularly Rubia Cordifolia), and Guggal (extracted from plantsin the genus Commiphora, particularly Commiphora Mukul), kola extract,chamomile, red clover extract, Piper methysticum, Bacopa monieriextract, sea whip, Glycyrrhiza glabra, mulberry, melaleuca (tea tree),mushroom extracts, Larrea divaricata, Rabdosia rubescens, Euglenagracilis, Fibraurea recisa Hirudinea, Chaparral Sorghum, sun flowerextract, Enantia chlorantha, Mitracarpe of Spermacocea genus, Buchubarosma, Lawsonia inermis L., Adiantium Capillus-Veneris L., Chelidoniummajus, Luffa cylindrical, Japanese Mandarin (Citrus reticulata Blancovar. unshiu), broccoli extract, Camelia sinensis, Imperata cylindrical,Glaucium Flavum, Cupressus Sempervirens, Polygonatum multiflorum,loveyly hemsleya, Sambucus Nigra, Phaseolus lunatus, Centaurium,Macrocystis Pyrifera, Turnera Diffusa, Anemarrhena asphodeloides,Portulaca pilosa, Humulus lupulus, Coffee Arabica, coffee berry, blackberry, Ilex Paraguariensis; and so on.

Oils and Lipids

The oil phase can contain any cosmetic or dermatological oil or amixture thereof. Examples of such oils include but are not limited toaliphatic hydrocarbons such as liquid paraffin, squalene, squalane,vaseline and ceresin; silicon oils such as dimethicone andcyclomethicones; vegetable oils such as avocado oil, apricot oil, almondoil, borage oil, borage seed oil, camellia oil, canola oil, castor oil,coconut oil, cocoa butter, corn oil, cottonseed oil, olive oil, eveningprimrose oil, flax seed oil, palm oil, palm kernel oil, peanut oil,rapeseed oil, safflower oil, sesame oil, sweet almond oil, rose hip oil,calendula oil, chamomile oil, eucalyptus oil, juniper oil, saffloweroil, sandalwood oil, tea tree oil, sunflower oil, soybean oil, wheatgerm oil; animal oils such as shark liver oil, cod liver oil, whale oil,beef tallow and butterfat; waxes such as beeswax, carnauba palm wax,spermaceti and lanolin; fatty acids such as lauric acid, myristic acid,palmitic, acid, stearic acid, oleic acid, behenic acid; omega-3 fattyacids such as alpha-linolenic acid, eicosapentaenoic acid, anddocosahexaenoic acid; omega-6 fatty acids such as linoleic acid andgamma-linolenic acid; aliphatic alcohols such as lauryl, stearyl, cetyl,and oleyl alcohol; and aliphatic esters such as isopropyl, isocetyl, oroctadecyl myristate, butyl stearate, hexyl laureate, diisopropyl esterof adipic acid, or diisopropyl sebacate; and/or mixtures thereof.Generally, the oils are refined and/or hydrogenated. Lipids includemonoglycerides, diglycerides, triglycerides, phospholipids, andceramides.

Suspending Agents

The compositions of the present invention may further contain asuspending agent, preferably at concentrations effective for suspendingwater-insoluble material in dispersed form in the compositions or formodifying the viscosity of the composition. Such concentrations canpreferably range from about 0.1% to about 10%, more preferably fromabout 0.25% to about 5.0%. Suspending agents useful herein includeanionic polymers and nonionic polymers. Useful herein are vinyl polymerssuch as cross linked acrylic acid polymers with the CTFA name Carbomer,cellulose derivatives and modified cellulose polymers such as methylcellulose, ethyl cellulose, nitro cellulose, sodium carboxymethylcellulose, crystalline cellulose, cellulose powder,polyvinylpyrrolidone, polyvinyl alcohol, guar gum, hydroxypropyl guargum, arabia gum, galactan, carob gum, pectin, agar, starch (rice, corn,potato, wheat), algae colloids (algae extract), microbiological polymerssuch as dextran, succinoglucan, pulleran, starch-based polymers such ascarboxymethyl starch, methylhydroxypropyl starch, alginic acid-basedpolymers such as sodium alginate, alginic acid propylene glycol esters,acrylate polymers such as sodium polyacrylate, polyethylacrylate,polyacrylamide, polyethyleneimine, and inorganic water soluble materialsuch as bentonite, aluminum magnesium silicate, laponite, hectonite, andanhydrous silicic acid. Actives aforementioned as thickening agents canalso be used herein as suspending agents.

Other optional suspending agents include crystalline suspending agentswhich can be categorized as acyl derivatives, long chain amine oxides,long chain acyl derivatives and mixtures thereof. These preferredsuspending agents include ethylene glycol esters of fatty acids, alkanolamides of fatty acids, long chain esters of long chain fatty acids(e.g., stearyl stearate, cetyl palmitate, etc.); long chain esters oflong chain alkanol amides (e.g., stearamide diethanolamide distearate,stearamide monoethanolamide stearate); and glyceryl esters (e.g.,glyceryl distearate, trihydroxystearin, tribehenin). Other suitablesuspending agents include primary amines having a fatty alkyl moietyhaving at least about 16 carbon atoms, examples of which includepalmitamine or stearamine, and secondary amines having two fatty alkylmoieties each having at least about 12 carbon atoms, examples of whichinclude dipalmitoylamine or di(hydrogenated tallow)amine. Still othersuitable suspending agents include di(hydrogenated tallow)phthalic acidamide, and crosslinked maleic anhydride-methyl vinyl ether copolymer.

Emulsifying Agents

Emulsifying agents include a wide variety of nonionic, cationic,anionic, zwitterionic, and amphoteric surfactants such as are known inthe art and discussed below. The hydrophilic surfactants (cationic,anionic, zwitterionic, amphoteric) useful herein can contain a singlesurfactant, or any combination of suitable surfactants. The exactsurfactant (or surfactants) chosen will depend upon the pH of thecomposition and the other components present.

Useful nonionic surfactants include the condensation products ofalkylene oxides with fatty acids (i.e., alkylene oxide esters of fattyacids), the condensation products of alkylene oxides with 2 moles offatty acids (i.e., alkylene oxide diesters of fatty acids), thecondensation products of alkylene oxides with fatty alcohols (i.e.,alkylene oxide ethers of fatty alcohols), the condensation products ofalkylene oxides with both fatty acids and fatty alcohols [i.e., whereinthe polyalkylene oxide portion is esterified on one end with a fattyacid and etherified (i.e., connected via an ether linkage) on the otherend with a fatty alcohol]. Nonlimiting examples of these alkylene oxidederived nonionic surfactants include ceteth-6, ceteth-10, ceteth-12,ceteareth-6, ceteareth-10, ceteareth-12, steareth-6, steareth-10,steareth-12, steareth-21, PEG-6 stearate, PEG-10 stearate, PEG-100stearate, PEG-12 stearate, PEG-20 glyceryl stearate, PEG-80 glyceryltallowate, PEG-10 glyceryl stearate, PEG-30 glyceryl cocoate, PEG-80glyceryl cocoate, PEG-200 glyceryl tallowate, PEG-8 dilaurate, PEG-10distearate, and mixtures thereof. Still other useful nonionicsurfactants include polyhydroxy fatty acid amide surfactants. Anespecially preferred surfactant corresponding to the above structure iscoconut alkyl N-methyl glucoside amide. Preferred among the nonionicsurfactants are those selected from the group consisting of steareth-21,ceteareth-20, ceteareth-12, sucrose cocotte, steareth-100, PEG-100stearate, and mixtures thereof. Other nonionic surfactants suitable foruse herein include sugar esters and polyesters, alkoxylated sugar estersand polyesters, C1-C30 fatty acid esters of C1-C30 fatty alcohols,alkoxylated derivatives of C1-C30 fatty acid esters of C1-C30 fattyalcohols, alkoxylated ethers of C1-C30 fatty alcohols, polyglycerylesters of C1-C30 fatty acids, C1-C30 esters of polyols, C1-C30 ethers ofpolyols, alkyl phosphates, polyoxyalkylene fatty ether phosphates, fattyacid amides, acyl lactylates, and mixtures thereof. Nonlimiting examplesof these emulsifiers include: polyethylene glycol 20 sorbitanmonolaurate (Polysorbate 20), polyethylene glycol 5 soya sterol,Steareth-20, Ceteareth-20, PPG-2 methyl glucose ether distearate,Ceteth-10, Polysorbate 80, cetyl phosphate, potassium cetyl phosphate,diethanolamine cetyl phosphate, Polysorbate 60, glyceryl stearate,polyoxyethylene 20 sorbitan trioleate (Polysorbate 85), sorbitanmonolaurate, polyoxyethylene 4 lauryl ether sodium stearate,polyglyceryl-4 isostearate, hexyl laurate, PPG-2 methyl glucose etherdistearate, PEG-100 stearate, and mixtures thereof. Another group ofnon-ionic surfactants useful herein are fatty acid ester blends based ona mixture of sorbitan or sorbitol fatty acid ester and sucrose fattyacid ester, the fatty acid in each instance being preferably C8-C24,more preferably C10-C20. The preferred fatty acid ester emulsifier is ablend of sorbitan or sorbitol C16-C20 fatty acid ester with sucroseC10-C16 fatty acid ester, especially sorbitan stearate and sucrosecocoate. This is commercially available from ICI under the trade nameArlatone 2121.

Also useful herein are cationic surfactants, especially dialkylquaternary ammonium compounds. Nonlimiting examples of these cationicemulsifiers include stearamidopropyl PG-dimonium chloride phosphate,behenamidopropyl PG dimonium chloride, stearamidopropyl ethyldimoniumethosulfate, stearamidopropyl dimethyl (myristyl acetate) ammoniumchloride, stearamidopropyl dimethyl cetearyl ammonium tosylate,stearamidopropyl dimethyl ammonium chloride, stearamidopropyl dimethylammonium lactate, and mixtures thereof. Especially preferred isbehenamidopropyl PG dimonium chloride. Nonlimiting examples ofquaternary ammonium salt cationic surfactants include those selectedfrom cetyl ammonium chloride, cetyl ammonium bromide, lauryl ammoniumchloride, lauryl ammonium bromide, stearyl ammonium chloride, stearylammonium bromide, cetyl dimethyl ammonium chloride, cetyl dimethylammonium bromide, lauryl dimethyl ammonium chloride, lauryl dimethylammonium bromide, stearyl dimethyl ammonium chloride, stearyl dimethylammonium bromide, cetyl trimethyl ammonium chloride, cetyl trimethylammonium bromide, lauryl trimethyl ammonium chloride, lauryl trimethylammonium bromide, stearyl trimethyl ammonium chloride, stearyl trimethylammonium bromide, lauryl dimethyl ammonium chloride, stearyl dimethylcetyl ditallow dimethyl ammonium chloride, dicetyl ammonium chloride,dicetyl ammonium bromide, dilauryl ammonium chloride, dilauryl ammoniumbromide, distearyl ammonium chloride, distearyl ammonium bromide,dicetyl methyl ammonium chloride, dicetyl methyl ammonium bromide,dilauryl methyl ammonium chloride, dilauryl methyl ammonium bromide,distearyl methyl ammonium chloride, distearyl methyl ammonium bromide,and mixtures thereof. Additional quaternary ammonium salts include thosewherein the C12 to C30 alkyl carbon chain is derived from a tallow fattyacid or from a coconut fatty acid. The term “tallow” refers to an alkylgroup derived from tallow fatty acids (usually hydrogenated tallow fattyacids), which generally have mixtures of alkyl chains in the C16 to C18range. The term “coconut” refers to an alkyl group derived from acoconut fatty acid, which generally have mixtures of alkyl chains in theC12 to C14 range. Examples of quaternary ammonium salts derived fromthese tallow and coconut sources include ditallow dimethyl ammoniumchloride, ditallow dimethyl ammonium methyl sulfate, di(hydrogenatedtallow) dimethyl ammonium chloride, di(hydrogenated tallow) dimethylammonium acetate, ditallow dipropyl ammonium phosphate, ditallowdimethyl ammonium nitrate, di(coconutalkyl)dimethyl ammonium chloride,di(coconutalkyl)dimethyl ammonium bromide, tallow ammonium chloride,coconut ammonium chloride, and mixtures thereof. An example of aquaternary ammonium compound having an alkyl group with an ester linkageis ditallowyl oxyethyl dimethyl ammonium chloride. More preferredcationic surfactants are those selected from behenamidopropyl PGdimonium chloride, dilauryl dimethyl ammonium chloride, distearyldimethyl ammonium chloride, dimyristyl dimethyl ammonium chloride,dipalmityl dimethyl ammonium chloride, distearyl dimethyl ammoniumchloride, stearamidopropyl PG-dimonium chloride phosphate,stearamidopropyl ethyldiammonium ethosulfate, stearamidopropyl dimethyl(myristyl acetate) ammonium chloride, stearamidopropyl dimethyl cetearylammonium tosylate, stearamidopropyl dimethyl ammonium chloride,stearamidopropyl dimethyl ammonium lactate, and mixtures thereof. Stillmore preferred cationic surfactants are those selected frombehenamidopropyl PG dimonium chloride, dilauryl dimethyl ammoniumchloride, distearyl dimethyl ammonium chloride, dimyristyl dimethylammonium chloride, dipalmityl dimethyl ammonium chloride, and mixturesthereof. A preferred combination of cationic surfactant and structuringagent is behenamidopropyl PG dimonium chloride and/or behenyl alcohol,wherein the ratio is preferably optimized to maintain or to enhancephysical and chemical stability, especially when such a combinationcontains ionic and/or highly polar solvents.

A wide variety of anionic surfactants can also be useful herein.Nonlimiting examples of anionic surfactants include the alkoylisethionates, and the alkyl and alkyl ether sulfates. The reactionproducts of fatty acids esterified with isethianonic acid andneutralized, i.e., the alkoyl isethionates typically have the formulaRCOOCH2CH2SO3M wherein R is alkyl or alkenyl of from about 10 to about30 carbon atoms, and M is a water-soluble cation such as ammonium,sodium, potassium and triethanolamine. For example, the fatty acids arederivated from coconut or palm kernel oil. Nonlimiting examples of theseisethionates include those alkoyl isethionates selected from ammoniumcocoyl isethionate, sodium cocoyl isethionate, sodium lauroylisethionate, sodium stearoyl isethionate, and mixtures thereof. Alsosuitable are salts of fatty acids, amids of methyl taurides. The alkyland alkyl ether sulfates typically have the respective formulae ROSO3Mand RO(C2H40)xSO3M, wherein R is alkyl or alkenyl of from about 10 toabout 30 carbon atoms, x is from about 1 to about 10, and M is awater-soluble cation such as ammonium, alkanolamines such astriethanolamine, monovalent metals, such as sodium and potassium, andpolyvalent metal cations such as magnesium and calcium. Preferably, Rhas from about 8 to about 18 carbon atoms, more preferably from about 10to about 16 carbon atoms, even more preferably from about 12 to about 14carbon atoms, in both the alkyl and alkyl ether sulfates. The alkylether sulfates are typically made as condensation products of ethyleneoxide and monohydric alcohols having from about 8 to about 24 carbonatoms. The alcohols can be synthetic or they can be derived from fats,e.g., coconut oil, palm kernel oil, tallow. Lauryl alcohol and straightchain alcohols derived from coconut oil or palm kernel oil arepreferred. Such alcohols are reacted with between about 0 and about 10,preferably from about 2 to about 5, more preferably about 3, molarproportions of ethylene oxide, and the resulting mixture of molecularspecies having, for example, an average of 3 moles of ethylene oxide permole of alcohol, is sulfated and neutralized. Another suitable class ofanionic surfactants are the water-soluble salts of the organic, sulfuricacid reaction products of the general formula R1-SO3-M, wherein R1 ischosen from the group including a straight or branched chain, saturatedaliphatic hydrocarbon radical having from about 8 to about 24,preferably about 10 to about 16, carbon atoms; and M is a cationdescribed hereinbefore. Still other anionic synthetic surfactantsinclude the class designated as succinamates, olefin sulfonates havingabout 12 to about 24 carbon atoms, and beta-alkyloxy alkane sulfonates.Examples of these materials are sodium lauryl sulfate and ammoniumlauryl sulfate. Other anionic surfactants suitable for use in thecompositions are the succinnates, examples of which include disodiumN-octadecylsulfosuccinnate; disodium lauryl sulfosuccinate; diammoniumlauryl sulfosuccinate; tetrasodiumN-(1,2-dicarboxyethyl)-N-octadecylsulfosuccinnate; diamyl ester ofsodium sulfosuccinic acid; dihexyl ester of sodium sulfosuccinic acid;and dioctyl esters of sodium sulfosuccinic acid. Other suitable anionicsurfactants include olefin sulfonates having about 10 to about 24 carbonatoms. In addition to the true alkene sulfonates and a proportion ofhydroxy-alkanesulfonates, the olefin sulfonates can contain minoramounts of other materials, such as alkene disulfonates depending uponthe reaction conditions, proportion of reactants, the nature of thestarting olefins and impurities in the olefin stock and side reactionsduring the sulfonation process. Another class of anionic surfactantssuitable for use in the compositions is the beta-alkyloxy alkanesulfonate class. Other anionic materials useful herein are soaps (i.e.,alkali metal salts, e.g., sodium or potassium salts) of fatty acids,typically having from about 8 to about 24 carbon atoms, preferably fromabout 10 to about 20 carbon atoms. The fatty acids used in making thesoaps can be obtained from natural sources such as, for instance, plantor animal-derived glycerides (e.g., palm oil, coconut oil, soybean oil,castor oil, tallow, lard, etc.) The fatty acids can also besynthetically prepared.

Amphoteric and zwitterionic surfactants are also useful herein. Examplesof amphoteric and zwitterionic surfactants which can be used in thecompositions of the present invention are those which are broadlydescribed as derivatives of aliphatic secondary and tertiary amines inwhich the aliphatic radical can be straight or branched chain andwherein one of the aliphatic substituents contains from about 8 to about22 carbon atoms (preferably C8-C18) and one contains an anionic watersolubilizing group, e.g., carboxy, sulfonate, sulfate, phosphate, orphosphonate. Examples are alkyl imino acetates, and iminodialkanoatesand aminoalkanoates of the formulas RN[CH2)mCO2M]2 and RNH(CH2)mCO2Mwherein m is from 1 to 4, R is a C8-C22 alkyl or alkenyl, and M is H,alkali metal, alkaline earth metal ammonium, or alkanolammonium.Preferred amphoteric surfactants for use in the present inventioninclude cocoamphoacetate, cocoamphodiacetate, lauroamphoacetate,lauroamphodiacetate, and mixtures thereof. Also included areimidazolinium and ammonium derivatives. Specific examples of suitableamphoteric surfactants include sodium 3-dodecyl-aminopropionate, sodium3-dodecylaminopropane sulfonate, N-alkyltaurines such as the oneprepared by reacting dodecylamine with sodium isethionate; N-higheralkyl aspartic acids; and the products sold under the trade name“Miranol”. Other examples of useful amphoterics include phosphates, suchas coamidopropyl PG-dimonium chloride phosphate (commercially availableas Monaquat PTC, from Mona Corp.). Zwitterionic surfactants suitable foruse in the composition are well known in the art, and include thosesurfactants broadly described as derivatives of aliphatic quaternaryammonium, phosphonium, and sulfonium compounds, in which the aliphaticradicals can be straight or branched chain, and wherein one of thealiphatic substituents contains from about 8 to about 18 carbon atomsand one contains an anionic group such as carboxy, sulfonate, sulfate,phosphate or phosphonate. Zwitterionics such as betaines are preferred.Examples of betaines include the higher alkyl betaines, such as cocodimethyl carboxymethyl betaine, lauryl dimethyl carboxymethyl betaine,lauryl dimethyl alphacarboxyethyl betaine, cetyl dimethyl carboxymethylbetaine, cetyl dimethyl betaine (available as Lonzaine 16SP from LonzaCorp.), lauryl bis-(2-hydroxyethyl) carboxymethyl betaine, stearylbis-(2-hydroxypropyl) carboxymethyl betaine, oleyl dimethylgamma-carboxypropyl betaine, laurylbis-(2-hydroxypropyl)alpha-carboxyethyl betaine, coco dimethylsulfopropyl betaine, stearyl dimethyl sulfopropyl betaine, lauryldimethyl sulfoethyl betaine, lauryl bis-(2-hydroxyethyl) sulfopropylbetaine, and amidobetaines and amidosulfobetaines (wherein theRCONH(CH2)3 radical is attached to the nitrogen atom of the betaine),oleyl betaine (available as amphoteric Velvetex OLB-50 from Henkel), andcocamidopropyl betaine (available as Velvetex BK-35 and BA-35 fromHenkel). Other useful amphoteric and zwitterionic surfactants includethe sultaines and hydroxysultaines such as cocamidopropylhydroxysultaine (available as Mirataine CBS from Rhone-Poulenc), and thealkanoyl sarcosinates corresponding to the formula RCON(CH3)CH2CH2CO2Mwherein R is alkyl or alkenyl of about 10 to about 20 carbon atoms, andM is a water-soluble cation such as ammonium, sodium, potassium andtrialkanolamine (e.g., triethanolamine), a preferred example of which issodium lauroyl sarcosinate.

Thickening Agents

Thickening agents suitable for inclusion in a composition describedherein include those agents commonly used as an excipient or a carrierfor topical application to increase the viscosity of the formulation.Thickening agents may also be used to improve the stability of theformulation and the product.

More specifically, such examples include but are not limited to,acrylamides copolymer, agarose, amylopectin, bentonite, calciumalginate, calcium carboxymethyl cellulose, carbomer, carboxymethylchitin, cellulose gum, dextrin, gelatin, hydrogenated tallow,hydroxylethyl-cellulose, hydroxypropylcellulose, hydroxypropyl starch,magnesium alginate, methylcellulose, microcrystalline cellulose, pectin,various polyethylene glycol's, polyacrylic acid, poly-methacrylic acid,polyvinyl alcohol, various polypropylene glycols, sodium acrylatescopolymer, sodium carrageenan, xanthan gum, and/or yeast beta-glucan.

More generally, carboxylic acid polymers useful thickening agents.Carboxylic acid polymers are cross-linked compounds containing one ormore monomers derived from acrylic acid, substituted acrylic acids, andsalts and esters of these acrylic acids and the substituted acrylicacids, wherein the cross-linking agent contains two or morecarbon-carbon double bonds and is derived from a polyhydric alcohol.Examples of commercially available carboxylic acid polymers usefulherein include the carbomers, which are homopolymers of acrylic acidcross-linked with allyl ethers of sucrose or pentaerytritol. Thecarbomers are available as the Carbopol® 900 series from B.F. Goodrich(e.g., Carbopol® 954). In addition, other suitable carboxylic acidpolymeric agents include copolymers of C10-30 alkyl acrylates with oneor more monomers of acrylic acid, methacrylic acid, or one of theirshort chain (i.e., C1-4 alcohol) esters, wherein the cross-linking agentis an allyl ether of sucrose or pentaerytritol. These copolymers areknown as acrylates/C10-30 alkyl acrylate crosspolymers and arecommercially available as Carbopol® 1342, Carbopol® 1382, Pemulen TR-1,and Pemulen TR-2, from B.F. Goodrich. Examples of preferred carboxylicacid polymer thickeners useful herein include those selected fromcarbomers, acrylates/C10-30 alkyl acrylate crosspolymers, and mixturesthereof.

Moreover, a wide variety of polysaccharides are useful herein asthickening agents. Non-limiting examples of polysaccharide gellingagents include those selected from cellulose, carboxymethylhydroxyethylcellulose, cellulose acetate propionate carboxylate,hydroxyethylcellulose, hydroxyethyl ethylcellulose,hydroxypropylcellulose, hydroxypropyl methylcellulose, methylhydroxyethylcellulose, microcrystalline cellulose, sodium cellulosesulfate, and mixtures thereof. Also useful herein are the alkylsubstituted celluloses. In these polymers, the hydroxy groups of thecellulose polymer is hydroxyalkylated (preferably hydroxyethylated orhydroxypropylated) to form a hydroxyalkylated cellulose which is thenfurther modified with a C10-30 straight chain or branched chain alkylgroup through an ether linkage. Typically these polymers are ethers ofC10-30 straight or branched chain alcohols with hydroxyalkylcelluloses.Examples of alkyl groups useful herein include those selected fromstearyl, isostearyl, lauryl, myristyl, cetyl, isocetyl, cocoyl (e.g.,alkyl groups derived from the alcohols of coconut oil), palmityl, oleyl,linoleyl, linolenyl, ricinoleyl, behenyl, and mixtures thereof.Preferred among the alkyl hydroxyalkyl cellulose ethers is the materialgiven the CTFA designation cetyl hydroxyethylcellulose, which is theether of cetyl alcohol and hydroxyethylcellulose. This material is soldunder the trade name Natrosol® CS Plus from Aqualon Corporation(Wilmington, Del.). Additional examples can be found in TheInternational Cosmetic Ingredient Dictionary and Handbook, the CosmeticBench Reference—Directory of Cosmetic Ingredients, the books provided bythe United States Pharmacopeia (USP) and the National Formulary (NF),and other references for cosmetic and pharmaceutical ingredients knownin the art. Other useful polysaccharides include scleroglucans which area linear chain of (1-3) linked glucose units with a (1-6) linked glucoseevery three units, a commercially available example of which isClearogel™ CS11 from Michel Mercier Products Inc. (Mountainside, N.J.).

Other thickening and gelling agents useful herein include materialswhich are primarily derived from natural sources. Non-limiting examplesof these gelling agent gums include acacia, agar, algin, alginic acid,ammonium alginate, amylopectin, calcium alginate, calcium carrageenan,carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guarhydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydratedsilica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp,locust bean gum, natto gum, potassium alginate, potassium carrageenan,propylene glycol alginate, sclerotium gum, sodium carboyxmethyl dextran,dextran sulfate, sodium carrageenan, tragacanth gum, xanthan gum, and/ormixtures thereof. In addition, the compositions of the present inventioncan also optionally contain polyacrylamide polymers, especially nonionicpolyacrylamide polymers including substituted branched or unbranchedpolymers. More preferred among these polyacrylamide polymers is thenonionic polymer given the CTFA designation polyacrylamide andisoparaffin and laureth-7, available under the trade name Sepigel 305from Seppic Corporation (Fairfield, N.J.). Other polyacrylamide polymersuseful herein include multi-block copolymers of acrylamides andsubstituted acrylamides with acrylic acids and substituted acrylicacids.

Preferred compositions of the present invention include a thickeningagent selected from carboxylic acid polymers, cross-linked polyacrylatepolymers, polyacrylamide polymers, and mixtures thereof, more preferablyselected from carboxylic acid polymers, polyacrylamide polymers, andmixtures thereof.

Penetration Enhancers

Penetration enhancers are the substances that facilitate the absorptionof penetrant through the skin or mucosal membranes by temporarilydiminishing the impermeability of the skin or, respectively, the mucosa.Ideally, these materials should be pharmacologically inert, nontoxic,nonirritating, non-allergenic, compatible with Octanoyl Carnosine,odorless, tasteless, colorless, and inexpensive and have good solventproperties. The enhancer should not lead to the significant loss of bodyfluids, electrolytes, and other endogenous materials, and skin or mucosashould regain its barrier properties on its removal within an acceptableperiod of time. No single penetration enhancer can possess all therequired properties. However, many enhancers exhibit many of theseattributes, and they have been described (for example as reviewed inDrug Development and Industrial Pharmacy 2000, 26, 1131-1140) or arebeing currently researched.

Anti-Histamines

Anti-histamines, also called histamine antagonists, are substances thatinhibit the action of histamine by blocking it from attaching tohistamine receptors; or by inhibiting the enzymatic activity ofhistidine decarboxylase, catalyzing the transformation of histidine intohistamine; or similar. Examples of anti-histamines are acrivastine,azelastine, brompheniramine, buclizine, bromodiphenhydramine,carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine,chlorodiphenhydramine, cimetidine, clemastine, cyproheptadine,desloratadine, dexbrom-pheniramine, deschlorpheniramine,dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine,doxylamine, ebastine, embramine, famotidine, fexofenadine, lafutidine,levocetirizine, loratadine, meclozine, mirtazapine, nizatidine,olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine,promethazine, pyrilamine, quetiapine, ranitidine, roxatidine,rupatadine, tripelennamine, and triprolidine.

The choice of additional substances to be included in the composition ismade depending on the constraints relating to Octanoyl Carnosine (or oneor more derivatives thereof) (e.g., stability, solubilization, etc.), ifenhanced and/or additional benefits and properties (e.g., anti-acne,anti-microbial, anti-wrinkle, skin lightening, anti-redness,antioxidant, skin protectant, sunscreen, hair growth, anti-inflammatory,emolliant, moisturization, enhanced skin penetration, etc.) of thecomposition are desired, and, where applicable, the use subsequentlyenvisaged for the composition.

As mentioned, the compositions of the invention may include one or moreadditional substances, various, conventional or not, which will providesome benefit to the object of the composition. More specifically, thecombination of Octanoyl Carnosine (or one or more derivatives thereof)with selected additional substances may lead to an enhanced efficacy ascompared to the use of Octanoyl Carnosine (or one or more derivativesthereof) alone. The enhanced efficacy can be additive (the sum ofefficacies of the individual agents alone), or it can be synergistic(larger than the sum of efficacies of the individual agents alone). Ofcourse, a decision to include an additional ingredient or substance andthe choice of a specific ingredient or substance depends on the specificuse of the composition and the product formulation.

For example, the use of one or more suitable anti-wrinkling substance(e.g., retinoic acid, retinol, transforming growth factor beta-1,selected peptides, etc.) will increase the clinical efficacy (e.g.,reduced skin wrinkles) of the composition containing Octanoyl Carnosine(or one or more derivatives thereof) after topical administration; theuse of one or more suitable emollient substance (e.g., octyldodecanol,etc.) will increase the clinical efficacy (e.g., improved skin feel orsensations) of the composition containing Octanoyl Carnosine (or one ormore derivatives thereof) after topical administration; the use of oneor more suitable humectant substance (e.g., glycerin, hyaluronic acid,etc.) will increase the clinical efficacy (e.g., increased skinmoisturization) of the composition containing Octanoyl Carnosine (or oneor more derivatives thereof) after topical administration; the use ofone or more suitable skin penetration enhancer substance (e.g.,propylene glycol, butylene glycol, ethanol, oleic acid, lauric acid,palmitic acid, isopropyl palmitate, DMSO, sodium lauryl sulfate, Azone®,etc.) for Octanoyl Carnosine (or one or more derivatives thereof) willincrease the clinical efficacy (e.g., reduced skin wrinkles) of thecomposition containing Octanoyl Carnosine (or one or more derivativesthereof) after topical administration; the use of one or more suitableanti-inflammatory substance (e.g., bisabolol, glycyrrhetinic acid,linoleic acid, borage seed oil, wheat germ oil, etc.) will increase theclinical efficacy (e.g., reduced irritation or redness of skin ormucosa) of the composition containing Octanoyl Carnosine (or one or morederivatives thereof) after topical administration; the use of one ormore suitable topical anesthetic substance (e.g., lidocaine, pramoxinehydrochloride, etc.) will increase the clinical efficacy (e.g., reducedlocal pain) of the composition containing Octanoyl Carnosine (or one ormore derivatives thereof) after topical administration; and the use ofone or more suitable topical anti-histamine substance (e.g.,diphenhydramine, etc.) will increase the clinical efficacy (e.g.,reduced local itch) of the composition containing Octanoyl Carnosine (orone or more derivatives thereof) after topical administration.

Carriers and Excipients

The compositions of the present invention can also contain one or morecarriers and/or excipients acceptable for a mode of administration(i.e., for topical application and/or for subcutaneous administration).Those skilled in the art will be able to routinely select an appropriatecarrier and/or excipient for the mode of administration. Depending inthe use and the way of administration, the compositions of the presentinvention can also contain a carrier and/or excipient acceptable forinjection, implantation, or subcutaneous placement.

The carrier and/or excipient can be in a wide variety of forms.Non-limiting examples of suitable carriers and/or excipients includesimple solutions (water or oil based), emulsions, dispersions,multi-phase systems, semi-solid forms, solid forms (powder, sticks,patches), skin masks, tissues, foams, and aerosols. For example,emulsion carriers and/or excipients can include, but are not limited to,oil-in-water, silicone-in-water, water-in-oil, water-in-silicone,water-in-oil-in-water, oil-in-water-in-oil, and oil-in-water-in-siliconeemulsions.

Depending upon the desired product form, preferred carriers and/orexcipients can contain an emulsion such as oil-in-water emulsions (e.g.,silicone-in-water) and water-in-oil emulsions, (e.g., water-in-siliconeemulsions). In one example, oil-in-water emulsions are especiallypreferred. Emulsions according to the present invention can contain anaqueous phase and a lipid or oil. Lipids and oils may be derived fromanimals, plants, or petroleum and may be natural or synthetic (e.g.,man-made).

Preferred emulsions can also contain a humectant, such as glycerin.Emulsions can further contain from about 0.1% to about 25%, morepreferably from about 0.2% to about 10%, of an emulsifying agent(emulsifier), based on the weight of the composition. Emulsifier agentsmay be nonionic, anionic, or cationic. Suitable emulsifiers aredisclosed for example in McCutcheon's Detergents and Emulsifiers, NorthAmerican Edition, pages 317-324 (1986).

Suitable emulsions may have a wide range of viscosities, depending onthe desired product form. The compositions of the present invention canbe in the form of pourable liquids, semi-solids, to highly viscoussystems (e.g., solids) under ambient conditions.

Any of the compositions can contain an aqueous carrier and/or excipient,which is typically present at a level of from about 20% to about 99%,preferably from about 60% to about 90%. The aqueous carrier and/orexcipient may contain water, or a miscible mixture of water and organicsolvent (e.g., alcohols, including but not limited to ethanol, glycerin,propylene glycol, butylene glycol, other glycols, etc.), but preferablycontain water with significant lower or no concentrations of organicsolvent, except as otherwise incidentally incorporated into thecomposition as minor ingredients of other essential or optionalcomponents.

The compositions of the subject invention, including but not limited tosolutions, lotions, serums and creams, may contain an acceptableemollient. Such compositions preferably contain from about 1% to about80% of the emollient. As used herein, “emollient” refers to a materialuseful for the prevention or relief of dryness, as well as for theprotection of the skin. A wide variety of suitable emollients is knownand may be used herein. In addition to the examples of emollientsprovided above, Sagarin, Cosmetics, Science and Technology, 2nd Edition,Vol. 1, pp. 32-43 (1972) contains numerous other examples of materialssuitable as an emollient. A preferred emollient is glycerin. Glycerin ispreferably used in an amount of from or about 0.001% to or about 80%,more preferably from or about 0.01% to or about 25%, still morepreferably from or about 0.1% to or about 10%, and even more preferablyfrom or about 2% to or about 5%.

Lotions, serums and creams according to the present invention generallycontain a carrier and/or excipient and one or more emollients. Lotionsand creams typically contain from about 1% to about 50%, preferably fromabout 1% to about 20%, of emollient; from about 50% to about 90%,preferably from about 60% to about 80%, water; and, optionally,additional substances in amounts sufficient to provide additionalbenefits. Creams are generally thicker than lotions and serums due tohigher levels of emollients, higher levels of thickeners, and/ordifferences in the emulsifying system.

Ointments of the present invention may contain a simple base of animalor vegetable oils or semi-solid hydrocarbons such as petrolatum;absorption ointment bases which absorb water to form emulsions; or watersoluble carriers, e.g., a water soluble solution carrier. Ointments mayfurther contain a thickening agent, such as described above and inSagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp.72-73 (1972), and/or an emollient. For example, an ointment may containfrom about 2% to about 10% of an emollient; from about 0.1% to about 2%of a thickening agent as well as one or more additional substances(s) inamounts sufficient to provide additional benefits.

Compositions of this invention useful for cleansing (“cleansers”) can beformulated with a suitable carrier, e.g., as described above, andpreferably contain from about 1% to about 30%, more preferably fromabout 5% to about 10%, of an acceptable surfactant. The surfactant issuitably selected from anionic, nonionic, zwitterionic, amphoteric andampholytic surfactants, as well as mixtures of these surfactants. Suchsurfactants are well known to those skilled in the detergency art.Examples of a broad variety of surfactants useful herein are describedabove and in McCutcheon's Detergents and Emulsifiers, North AmericanEdition (1986), published by Allured Publishing Corporation. Thecleansing compositions can optionally contain, at their art-establishedlevels, other materials which are conventionally used in cleansingcompositions.

As used herein, the term “foundation” refers to a liquid, semi-liquid,semi-solid, or solid skin cosmetic which includes, but is not limited tolotions, creams, gels, pastes, cakes, and the like. Typically thefoundation is used over a large area of the skin, such as over the face,to provide a particular look. Foundations are typically used to providean adherent base for color cosmetics such as rouge, blusher, powder andthe like, and tend to hide skin imperfections and impart a smooth, evenappearance to the skin. Foundations of the present invention include adermatologically acceptable carrier and may include conventionalingredients such as oils, colorants, pigments, emollients, fragrances,waxes, stabilizers, and the like.

The compositions of the present invention can also contain a liquid thatis acceptable for injection to and/or under the skin if the compositionis to be injected. Any suitable acceptable liquid as known in the art orotherwise can be used.

Composition Preparation

The compositions of the present invention are generally prepared byconventional methods such as are known in the art of making compositionssuitable for topical application. Such methods can typically beconducted in one or more steps, with or without heating, cooling, andthe like.

In addition, the compositions of the present invention can also beprepared by conventional methods such as are known in the art of makingcompositions suitable for injections.

As used herein, a “formulation” is a mixture prepared according to aspecific procedure.

The physical form of the compositions according to the invention is notimportant. They may be in any galenic form such aerosols, creams,lotions, milk or cream ointments, gels, emulsions, dispersions,solutions, suspensions, cleansers, foundations, anhydrous preparations(sticks, in particular lip balm, body and bath oils), shower and bathgels, shampoos and scalp treatment lotions, cream or lotion for care ofskin or hair, gel or solution for care of skin or hair, cream or lotionfor care of the genitals (e.g., vulva, vagina, penis, scrotum), gel orsolution for care of genitals, make-up removing lotions or creams,sunscreen lotions, milks, artificial suntan lotions; pre-shave, shave orafter shave creams, foams, gels or lotions; make-up, lipsticks, mascarasor nail varnishes; skin essences, serums; adhesive or absorbentmaterials, skin masks; tissues; hydrating patches, transdermal patches,iontophoretic patches, microneedle patches; powders; emollient lotion,sprays, oils for the body and the bath, foundation tint bases, pomade,colloid, compact or solid suspension, pencil, sprayable or brossableformulation, blush, rouge, eyeliner, lip liner, lip gloss, facial orbody powder, mousse or styling gels, nail conditioner, lip balms, skinconditioners, anorectal creams, hygiene cream, moisturizers, hairsprays, hair conditioners, soaps, body exfoliants, astringents,depilatories and permanent waving solutions, anti-dandruff formulations,anti-hair loss formulations, anti-sweat and anti-perspirantformulations, nose sprays; and so on.

These compositions can also be presented in the form of lipsticksintended to apply color or to protect the lips from cracking, or ofmake-up products for the eyes or tints and tint bases for the face.Compositions in accordance with the invention include cosmetics,personal care products, feminine products, male products, hygieneproducts, and dermatological or pharmaceutical preparations.

The compositions of the present invention may also be applied on animalskin when wounds or defects or disorders of animal skin affecting theextracellular matrix are present.

The compositions according to the present invention may be prepared inthe form of solution, dispersion, emulsion, paste, or powder,individually or as a premix or in vehicles individually or as a premixin vectors such as macro-, micro-, or nanocapsules, macro-, micro- or,nanospheres, liposomes, oleosomes, cubosomes; macro-, micro-, ornanoparticles; or macro-, micro or nanosponges; or macro-, micro-, andnanocapsules; or macro-, micro- or nanospheres; micro- ornano-emulsions; or adsorbed onto tip of needles; or adsorbed ontomicroneedles or onto microneedle arrays; or adsorbed to organic polymerpowders, talcs, bentonites, or other inorganic or organic supports.

Furthermore, the compositions according to the present invention may beused in any form whatsoever, in a form bound to or incorporated in orabsorbed in or adsorbed on macro-, micro-, and nanoparticles; or macro-,micro or nanosponges; or macro-, micro-, and nanocapsules; or macro-,micro- or nanospheres; or adsorbed (e.g., by coating) onto microneedlepatches or arrays (such as described by Ameri M. et al., Pharm Res 2010,27: 303-313); for the treatment of textiles, natural or syntheticfibers, wools, and any materials that may be used for clothing orunderwear for day or night intended to come into contact with the skin,handkerchiefs or cloths, to exert their effect via this skin/textilecontact and to permit continuous topical deliver.

The compositions according to the present invention may also be preparedor used in a form of a device (e.g., medical device, combination betweendrug and medical device). Preferred devices include, but are not limitedto, devices for overcoming biological barriers such as ultrasounddevices (i.e., sonophoresis, sonoporation, acoustic ablation), electricdevices (iontophoresis, electroporation), high pressure devices (i.e.,liquid injection, powder injection), microneedles (i.e., solid, hollow,degradable, coated), thermal and optical devices (i.e., light, infrared,laser, radio-frequency), other physical devices reducing the skinbarrier (i.e., plasma devices, micro-dermabrasion, dermabrasion, suctiondevices, macro-needle devices, etc.), devices reducing the skin barrierby chemical means (i.e., chemical exfoliating devices, skin corrosion(e.g., using NaOH) devices), and/or any combination or combinationdevice thereof. Some example of methods and devices for overcomingbiological barriers have been described in Advanced Drug DeliveryReviews 2013, 65, 100-103 (incorporated herein as reference).

In addition, the compositions according to the present invention may beused in any form intended to be placed into the skin or mucosal tissue,or under the skin or mucosal tissue (e.g., by injection, implantation,or subcutaneous placement).

Method of Treatment

The present invention concerns compositions for their application as acosmetic, personal care, or a medicinal product.

The composition according to the invention can be applied topically ontoany areas of the face, neck, neckline, décolleté, scalp, hand, palm,arm, leg, foot, sole, chest, breast, back, abdomen, buttock, vulva, orpenis and scrotum, anus, and/or any other skin areas of the human body.

Further, the composition according to the invention can be also appliedlocally or topically onto any areas of the eye, mouth, nose; breastnipples, vulva, vagina and introitus; or penis and scrotum; rectum,and/or any other mucosal areas of the human body.

Furthermore, the composition according to the invention can also beapplied locally or topically to other surfaces of the human body,including hair and nail, or any wound, scar, or skin and mucosal surfaceareas affected by atrophy, or other conditions, disorders and diseasesassociated with changes in extracellular matrix components.

In addition, the compositions according to the present invention mayalso be applied by injection, implantation, or subcutaneous placement.

For example, the compositions described herein can be applied using asyringe, a micro-cannula, a patch, an iontophoretic patch, microneedles,and/or a microneedle array or patch. In addition, the composition can bealso applied in conjunction (i.e., before, after, or simultaneously)with the use of other skin devices changing the penetrationcharacteristics of skin such as, for example, laser, light, infrared,radiofrequency, ultrasound, electroporation, sonophoresis, thermal,plasma, and/or high pressure devices, and/or any combination(s)(including combination devices) thereof. Any other commonly used meansof administration can also be utilized.

In addition, the compositions according to the present invention mayalso be applied in animals.

In one example, the present invention concerns cosmetic treatmentmethods to improve the general state of the skin involving topicalapplication of an effective amount of the composition as defined aboveto the skin. More specifically, these methods can be used to preventand/or treat the signs of intrinsic and extrinsic skin aging; to preventand/or treat skin slackening and/or improve tone and/or firmness and/orelasticity of the skin; to prevent and/or treat skin atrophy and/orimprove the density of the dermis and epidermis; to give or returnvolume to the dermis and epidermis; to prevent and/or treat skindehydration; to prevent and/or treat skin roughness; to prevent and/ortreat cellulite, to prevent and/or treat stretch marks, to reduceexpansion and/or prevent the development of adipose tissue within thehypodermis; to lighten and/or whiten the skin; to prevent and/or treatglycation of molecules in the skin; and/or to prevent and/or treatdegradation of the skin due to the effects of oxidation.

The present invention also provides methods to improve the general stateof the atrophic tissue involving topical application of an effectiveamount of the composition as defined above to the tissue. Morespecifically, such methods can be used to restore damaged skin; torestore skin after cosmetic and dermatological procedures; to preventand/or treat atrophy of the female genitals, to prevent and/or treatvulvovaginal atrophy; to prevent and/or treat skin conditions anddisorders related to menopause; to prevent and/or treat skin conditionsand disorders associated with reduced estrogen levels in females; toprevent and/or treat vulvodynia; to prevent and/or treat vulvar lichensclerosus; to prevent and/or treat vulvar dermatoses; to prevent and/ortreat the signs of intrinsic and extrinsic aging of the female genitals;and/or to prevent and/or treat the signs of intrinsic and extrinsicaging of male genitals.

Some benefits (e.g., moisturization, soothing, calming, tightening,smooth feel, etc.) can be noticed within a few hours to a few days aftertopically applying the compositions according to the present inventionon the affected human skin or human tissue (for example vulva and/orvagina). However, it takes generally at least 30 days to notice benefits(e.g., anti-aging, wrinkle reducing, skin lightening, anti-redness,improving atrophy, wound healing, etc.). Thereby, the composition shouldbe applied to the affected human skin or human tissue at least once totwice a day.

Determination of an effective dose (e.g., therapeutically, cosmetically,pharmaceutically, and/or medicinally effective dose) of any of thecompositions of the instant invention is within the routine level ofskill in the art.

Kits and Dosage Forms

According to the invention, products or devices with severalcompartments or kits (having one or more containers) may be proposed toapply the compositions of the invention. By way of non-limiting example,a first compartment or container having a composition including theOctanoyl Carnosine, the selected tri-peptides, and/or tetra-peptides (orone or more derivatives thereof), and one or more additional substances(e.g., one or more biologically active ingredients and/or one or moreinactive ingredients such as an excipient and/or a carrier) in a secondcompartment or container, the compositions contained in the said firstand second compartments in this case being considered to be acombination composition for simultaneous, separate or step-wise use intime, particularly in any one of the treatments defined above.Alternatively, kits according to the invention may include thecomponents of the compositions in separate compartments or containers orcertain components can be in the same compartments or containers whileothers are in separate compartments or containers. Such kits will alsopreferably include instructions for use.

Any of the compositions described herein may be supplied in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of Octanoyl Carnosine (and one ormore additional active ingredients) calculated to produce the desiredcosmetic, personal care or therapeutic effect in association with therequired cosmetic and/or pharmaceutical carrier(s). The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the compositions and theparticular maintenance, therapeutic or prophylactic effect to beachieved, and (b) the limitations inherent in the art of compoundingOctanoyl Carnosine (and one or more additional active ingredients) forthe treatment of individuals.

The unit dosage form is any of a variety of forms, including, forexample, but not limited to, a solution, any semi-solid form, a capsule,a bag, a tablet, a single pump on an aerosol or a vial. The quantity ofactive ingredient(s) in a unit dose of composition is an effectiveamount and is varied according to the particular treatment involved.

One skilled in the art will appreciate that it is sometimes necessary tomake routine variations to the dosage depending on the age and conditionof the patient. The dosage will also depend on the route ofadministration.

EXAMPLES

Examples of methods of chemical synthesis of Octanoyl Carnosine,Palmitoyl-GHK, and GEKG (SEQ ID NO: 1), as well as suitable compositionsand their preparation according to the present invention will bedescribed hereafter. These compositions and their preparation arerepresentative, but do not restrict, the scope of the invention.

The Examples set forth herein are meant to exemplify the various aspectsof carrying out the invention and are not intended to limit theinvention in any way. Unless otherwise specified, it is to be understoodthat the concentrations of the ingredients in the compositions of theinvention are in weight percentages (w %), based on the total weight ofthe composition. All measurements are performed at 25° Celsius unlessstated otherwise.

The following examples describe and demonstrate various aspects withinthe scope of the present invention. The examples are only given forillustrative purposes and should not be considered to be restrictive tothis invention. Additionally for illustrative purposes severalcompositions and their use for topical administration will be described.

Example 1: Example of Synthesis of Peptide Octanoyl Carnosine

The di-peptide Octanoyl Carnosine can be prepared according to standardprocedures in peptide chemistry using solid-phase synthesis orliquid-phase synthesis. As an example, one way of the synthesis andpurification of Octanoyl Carnosine (Octanoyl-beta-Ala-His-OH) in itsacetate salt form by solid-phase synthesis is illustrated in FIG. 1.

The Octanoyl Carnosine synthesized can be purified using standardmethods (e.g., crystallization (as shown in this Example), thin layerchromatography, column chromatography, preparative HPLC, liquid-liquidextraction, etc.). Its identity can be confirmed by ESI-MS, and itspurity can be determined by HPLC. Other analytical methods for peptidesor lipoamino-peptides known in the art can also be used to determine theidentity and purity.

Example 2: Example of Synthesis of Palmitoyl-GHK

The tri-peptide Palmitoyl-GHK can be prepared according to standardprocedures in peptide chemistry using solid-phase synthesis orliquid-phase synthesis. As an example, one way of the synthesis andpurification of Palmitoyl-GHK (Palmitoyl-Gly-His-Lys-OH) in its acetatesalt form by solid-phase synthesis is illustrated in FIG. 2.

The Palmitoyl-GHK synthesized can be purified using standard methods(e.g., crystallization (as realized in this Example), thin layerchromatography, column chromatography, preparative HPLC, liquid-liquidextraction, etc.). Its identity can be confirmed by ESI-MS, and itspurity can be determined by HPLC. Other analytical methods for peptidesor lipoamino-peptides known in the art can also be used to determine theidentity and purity.

Example 3: Example of Synthesis of Peptide GEKG (SEQ ID NO: 1)

Peptide GEKG (SEQ ID NO: 1) (NH.sub.2-Gly-Glu-Lys-Gly-OH) (SEQ ID NO: 1)can be prepared according to standard procedures in peptide chemistryusing solid-phase synthesis or liquid-phase synthesis known in the art.(See, e.g., Merrifield, J. AM. CHEM. SOC. 85:2149-54 (1963)(incorporated herein by reference)).

The GEKG (SEQ ID NO: 1) synthesized can be purified using standardmethods (e.g., crystallization (as realized in this Example), thin layerchromatography, column chromatography, preparative HPLC, liquid-liquidextraction, etc.). Its identity can be confirmed by ESI-MS, and itspurity can be determined by HPLC. Other analytical methods for peptidesor lipoamino-peptides known in the art can also be used to determine theidentity and purity.

Example 4: Preparation of Compositions

This Example illustrates the preparation of a series of compositionssuitable for topical application in accordance to the present invention.These compositions may contain Octanoyl Carnosine (or one or morederivatives thereof) alone in a carrier acceptable for topicalapplications; the combination of both Octanoyl Carnosine and GEKG (SEQID NO: 1) (or one or more derivatives thereof); the combination of bothOctanoyl Carnosine and Palmitoyl-GHK (or one or more derivativesthereof); and/or the combination of Octanoyl Carnosine, GEKG (SEQ IDNO: 1) and Palmitoyl-GHK (or one or more derivatives thereof). Inaddition, any of the compositions may also include one or moreadditional substances and/or one or more acceptable carriers and/orexcipients suitable for topical applications.

The peptides Octanoyl Carnosine, Palmitoyl-GHK, GEKG (SEQ ID NO: 1) (aswell as their derivatives) can be synthesized in accordance with themethods described in Examples 1, 2 and 3, respectively. Alternatively,they can be obtained by different processes of synthesis andpurifications known in the art. Octanoyl Carnosine, Palmitoyl-GHK, GEKG(SEQ ID NO: 1) (or their derivatives) may be also obtained from anypeptide manufacturer or supplier; either in solid form (i.e., as salt athigh purity; e.g., larger than 75%, ideally larger than 90%, and moreideally larger than 95%), or already solublized in a simple andacceptable solvent, excipient, and/or carrier suitable for topicalapplication (e.g., water, glycerin, propylene glycol, butylene glycol,hexylene glycol, low molecular weight PEGs such as PEG-400, and/or anymixtures thereof; with or without suitable surfactants helping tosolubilize and stabilize the peptides; and/or with or withoutanti-microbial preservatives; and/or with or without chemicalstabilizers including antioxidants or iron chelators).

Preferably, Octanoyl Carnosine (or one or more derivatives thereof) ispresent in the compositions in proportions of between 0.0001% and 10% ofthe total weight of the composition, more preferably between 0.001% and5%, and even more preferably between 0.005% and 1%, depending on the useof the composition and the more or less potent effect sought. Thus, oneof the preferred composition contains Octanoyl Carnosine (or one or morederivatives thereof) in proportions of between 0.005% and 1% in acarrier acceptable for topical application.

The compositions according to the invention can be prepared by addingOctanoyl Carnosine (or one or more derivatives thereof), either in itssolid form, or solubilized in a suitable solvent, excipient, or carriersuitable for topical application, at any stage of the preparationprocess of the composition. Typically, however, Octanoyl Carnosine (orone or more derivatives thereof) is added as part of the aqueous phaseduring the preparation process of the composition. For instance in caseof oil-in-water type compositions, hydrogels, and aqueous systems,Octanoyl Carnosine (or one or more derivatives thereof) can be added asthe last step to the previously prepared bulk of the composition bymixing or homogenizing (e.g., using a mixer or homogenizer) OctanoylCarnosine (or one or more derivatives thereof), which has beenpreviously solubilized or incorporated into a solvent, excipient, and/orcarrier system, to the bulk of the composition at lower temperatures(e.g., typically below 60° C.; and ideally between about 20° to 40° C.).

For example, a solvent, excipient, and/or carrier system suitable fortopical application and able to solubilize or incorporate OctanoylCarnosine (or one or more derivatives thereof) can contain one or moreof the following ingredients: water, glycerin, propylene glycol,propanediol, butylene glycol, hexylene glycol, low molecular weight PEGssuch as PEG-400, cyclodextrins, ethanol, and/or any mixtures thereof.Additionally, this solvent or carrier system may further containsuitable surfactants (e.g., Tween 20, Tween 80, etc.) to help solubilizeand stabilize the peptides, and/or anti-microbial preservatives to helpprevent microbial growth and contamination, and/or antioxidants or ironchelators (e.g., EDTA, etc.) as chemical stabilizers. Water, glycerin,propylene glycol, propanediol, butylene glycol, cyclodextrins, and/orany mixtures thereof, are the preferred ingredients.

In most instances, the additional ingredients will include a cosmetic,dermatologically or pharmaceutically acceptable carrier either alone orin combination with other additional ingredients. The amounts ofadditional ingredients may range from about 90% to about 99.9999%,preferably from about 95% to about 99.999%, more preferably from about99% to about 99.999%, of the composition. In short, the percentage ofadditional ingredients in the compositions of the invention depends onthe percentages of Octanoyl Carnosine, Palmitoyl GHK, and/or GEKG (SEQID NO: 1) present in a given composition (i.e., the additionalingredients make up the balance of the composition). If carriers (eithersingularly, such as water, or complex co-solvents) are used, they maymake up the entire balance of the compositions.

When preparing compositions combining peptide Octanoyl Carnosine (or oneor more derivatives thereof) with other peptides, including but notlimited to peptide Palmitoyl-GHK (or one or more derivatives thereof)and/or peptide GEKG (SEQ ID NO: 1) (or one or more derivatives thereof),those peptides can be adding either in their solid form, or solubilizedin a suitable solvent, excipient, and/or carrier for topicalapplication, at any stage of the composition preparation process. Often,the peptides can be added as part of the aqueous phase during thepreparation process of the composition. For instance in case ofoil-in-water type compositions, hydrogels, and aqueous systems, thepeptides can be added as the last step to the previously prepared bulkof the composition by mixing the peptides, which have been previouslyincorporated into a solvent, excipient, and/or carrier for topicalapplication, to the bulk of the composition at lower temperatures (e.g.,typically below 60.degree. C.; and ideally between about 20.degree. to40.degree. C.). For example, a solvent, excipient, and/or carriersuitable to incorporate the peptides into the composition can includeone or more of the following ingredients: water, glycerin, propyleneglycol, propanediol, butylene glycol, hexylene glycol, low molecularweight PEGs such as PEG-400, cyclodextrins, ethanol, and/or any mixturesthereof. Additionally, this solvent, excipient and/or carrier mayfurther contain suitable surfactants (e.g., Tween 20, Tween 80, etc.) tohelp solubilize and stabilize the peptides, and/or anti-microbialpreservatives to help prevent microbial growth and contamination, and/orantioxidants or iron chelators (e.g., EDTA, etc.) as chemicalstabilizers. Water, glycerin, propylene glycol, propanediol, butyleneglycol, cyclodextrins, and/or any mixtures thereof are the preferredingredients.

However, when preparing compositions combining peptides which are not orpoorly water soluble, or if the peptides cannot be solubilized asdescribed above using suitable solvents, excipients, and/or carrierknown to the person of skill in the art, the peptides can be added aspart of the oil phase during the preparation process of the composition.Thereby, the peptides can be either added to the oil phase in theirsolid form, or solubilized in a suitable solvent or carrier system(e.g., oils) suitable for topical application and able to solubilize orincorporate the peptides.

Alternatively, instead of at the end of the preparation process of anoil-in-water composition for example, Octanoyl Carnosine (or one or morederivatives thereof) may be added already at an earlier step in theprocess. Thereby, Octanoyl Carnosine (or one or more derivativesthereof) can be added when preparing the water phase of the composition;either in its solid form, or solubilized in a suitable solvent orcarrier system for Octanoyl Carnosine (or one or more derivativesthereof).

When combining Octanoyl Carnosine (or one or more derivatives thereof)with both Palmitoyl-GHK (or one or more derivatives thereof) and GEKG(SEQ ID NO: 1) (or one or more derivatives thereof), the optimal weightratio of Octanoyl Carnosine (or one or more derivatives thereof) toPalmitoyl-GHK (or one or more derivatives thereof) to GEKG (SEQ IDNO: 1) (or one or more derivatives thereof) is 4 parts OctanoylCarnosine (or one or more derivatives thereof): 1 part Palmitoyl-GHK (orone or more derivatives thereof): 5 parts GEKG (SEQ ID NO: 1) (or one ormore derivatives thereof) (parts refer to parts per weight), asdiscovered during in vitro studies for hyaluronic acid formation (seeExample 5, infra). Thus, one of the preferred composition containsOctanoyl Carnosine (or one or more derivatives thereof), Palmitoyl-GHK(or one or more derivatives thereof) and GEKG (SEQ ID NO:1) (or one ormore derivatives thereof) at a weight ratio of 4:1:5; whereas OctanoylCarnosine (or one or more derivatives thereof) is present in thecompositions in proportions between 0.001% and 1%, in a carrier orexcipient acceptable for topical application.

In accordance to the present invention, the following illustratessuitable compositions for topical applications containing either 0.01%,0.013%, 0.02%, or 0.1% of the combination of Octanoyl Carnosine,Palmitoyl-GHK and GEKG (SEQ ID NO: 1) (or one or more derivativesthereof) at a weight ratio of 4:1:5.

Furthermore, also in accordance to the present invention, the followingalso illustrates suitable compositions for topical applicationscontaining either 0.01%, 0.1%, 0.25%, 0.5%, 1%, or 5% of OctanoylCarnosine (or one or more derivatives thereof).

Provided herein is information on the preparation of such exemplarycompositions. However, the person of skill in the art will understandthat any other suitable methods can also be used to prepare compositionsin accordance with the instant invention.

The compositions obtained as described in the following were tested forphysicochemical stability (including, but not limited to, changes incolor, odor, viscosity, pH, and/or appearance) under acceleratedconditions (40° C. to 50° C.) for up to three to six months. During thisperiod, the compositions were stable; meaning that the color, odor,viscosity, pH, and the appearance did not change, or only changed to alimited and acceptable extent (±10% from baseline). If indicated,testing for chemical stability of the drug actives (e.g., OTC drugactives such as sunscreen actives, hydroquinone, retinoic acid, acnedrug active, rosacea drug active, etc.) in the compositions wereadditionally realized by acceptable analytical methods (e.g., by HPLC,etc.).

The local tolerability (irritant and allergic contact dermatitispotential) of the compositions provided herein was determined by patchtests with challenge (repeated insult patch test) on the back of humans,and, during in use tests for few of the compositions. Those testsrevealed that these compositions are of acceptable local tolerability.This means that the compositions intended for cosmetic, feminine,personal care or hygiene use are neither contact irritants nor contactallergens. The compositions intended for pharmaceutical use (i.e.,medicaments) are of a negligible to minor potential for inducingirritant and allergic contact dermatitis in humans. Accordingly, thosecompositions are suitable for topical use since they were shown to bestable and of acceptable local tolerability.

The compositions can be filled into suitable packaging (containers) suchas, for example, tubes, pumps, airless pumps, jars, bottles, pens,aerosol containers, or other containers depending on use andadministration. The compositions are generally commercialized in thosecontainers.

Composition A1: Example of a Serum Containing 0.1% of Octanoyl Carnosine

A serum containing 0.1% of Octanoyl Carnosine containing high levels ofhyaluronic acid was prepared as described above and contained thefollowing other components in order of descending predominance: WATER,SODIUM HYALURONATE, CITRIC ACID, PHENOXYETHANOL, SODIUM METHYLPARABEN,SODIUM BUTYLPARABEN, SODIUM PROPYLPARABEN, and SODIUM CHLORIDE.

Composition A2: Example of a Serum Containing 0.25% of OctanoylCarnosine

A serum containing 0.25% of Octanoyl Carnosine containing high levels ofhyaluronic acid was prepared as described above and contained thefollowing other components in order of descending predominance: WATER,SODIUM HYALURONATE, CITRIC ACID, PHENOXYETHANOL, SODIUM METHYLPARABEN,SODIUM BUTYLPARABEN, SODIUM PROPYLPARABEN, and SODIUM CHLORIDE.

Composition A3: Example of a Serum Containing 0.5% of Octanoyl Carnosine

A serum containing 0.5% of Octanoyl Carnosine containing high levels ofhyaluronic acid was prepared as described above and contained thefollowing other components in order of descending predominance: WATER,SODIUM HYALURONATE, CITRIC ACID, PHENOXYETHANOL, SODIUM METHYLPARABEN,SODIUM BUTYLPARABEN, SODIUM PROPYLPARABEN, and SODIUM CHLORIDE.

Composition A4: Example of a Serum Containing 1% of Octanoyl Carnosine

A serum containing 1% of Octanoyl Carnosine containing high levels ofhyaluronic acid was prepared as described above and contained thefollowing other components in order of descending predominance: WATER,SODIUM HYALURONATE, CITRIC ACID, PHENOXYETHANOL, SODIUM METHYLPARABEN,SODIUM BUTYLPARABEN, SODIUM PROPYLPARABEN, and SODIUM CHLORIDE.

Composition A5: Example of a Serum Containing 5% of Octanoyl Carnosine

A serum containing 5% of Octanoyl Carnosine containing high levels ofhyaluronic acid was prepared as described above and contained thefollowing other components in order of descending predominance: WATER,SODIUM HYALURONATE, CITRIC ACID, PHENOXYETHANOL, SODIUM METHYLPARABEN,SODIUM BUTYLPARABEN, SODIUM PROPYLPARABEN, and SODIUM CHLORIDE.

Composition B1: Example of Oil-in-Water Cream Containing 0.01% ofOctanoyl Carnosine

An oil-in-water cream containing 0.01% Octanoyl Carnosine, free ofparabens and triethanolamine, was prepared as described above andcontained the following other components in order of descendingpredominance: water, octyldodecanol, decyl oleate, glyceryl stearate,propanediol, glycerin, stearic acid, Triticum vulgare (wheat) germ oil,ceteareth-20, cetyl alcohol, Borago officinalis (borage) seed oil,dimethicone, Triticum vulgare (wheat) seed extract, myreth-3 myristate,ceteareth-12, tocopheryl acetate, tocopherol, cetearyl alcohol, cetylpalmitate, carbomer, disodium EDTA, sodium hydroxide, phenoxyethanol,ethylhexylglycerin, hexylene glycol, and caprylyl glycol.

Composition B2: Example of Oil-in-Water Cream Containing 0.1% ofOctanoyl Carnosine

An oil-in-water cream containing 0.1% Octanoyl Carnosine, free ofparabens and triethanolamine, was prepared as described above andcontained the following other components in order of descendingpredominance: water, octyldodecanol, decyl oleate, glyceryl stearate,propanediol, glycerin, stearic acid, Triticum vulgare (wheat) germ oil,ceteareth-20, cetyl alcohol, Borago officinalis (borage) seed oil,dimethicone, Triticum vulgare (wheat) seed extract, myreth-3 myristate,ceteareth-12, tocopheryl acetate, tocopherol, cetearyl alcohol, cetylpalmitate, carbomer, disodium EDTA, sodium hydroxide, phenoxyethanol,ethylhexylglycerin, hexylene glycol, and caprylyl glycol.

Composition B3: Example of Oil-in-Water Cream Containing 0.5% ofOctanoyl Carnosine

An oil-in-water cream containing 0.5% Octanoyl Carnosine, free ofparabens and triethanolamine, was prepared as described above andcontained the following other components in order of descendingpredominance: water, octyldodecanol, decyl oleate, glyceryl stearate,propanediol, glycerin, stearic acid, Triticum vulgare (wheat) germ oil,ceteareth-20, cetyl alcohol, Borago officinalis (borage) seed oil,dimethicone, Triticum vulgare (wheat) seed extract, myreth-3 myristate,ceteareth-12, tocopheryl acetate, tocopherol, cetearyl alcohol, cetylpalmitate, carbomer, disodium EDTA, sodium hydroxide, phenoxyethanol,ethylhexylglycerin, hexylene glycol, and caprylyl glycol.

Composition B4: Example of Oil-in-Water Cream Containing 1% of OctanoylCarnosine

An oil-in-water cream containing 1% Octanoyl Carnosine, free of parabensand triethanolamine, was prepared as described above and contained thefollowing other components in order of descending predominance: water,octyldodecanol, decyl oleate, glyceryl stearate, propanediol, glycerin,stearic acid, Triticum vulgare (wheat) germ oil, ceteareth-20, cetylalcohol, Borago officinalis (borage) seed oil, dimethicone, Triticumvulgare (wheat) seed extract, myreth-3 myristate, ceteareth-12,tocopheryl acetate, tocopherol, cetearyl alcohol, cetyl palmitate,carbomer, disodium EDTA, sodium hydroxide, phenoxyethanol,ethylhexylglycerin, hexylene glycol, and caprylyl glycol.

Composition B5: Example of Oil-in-Water Cream Containing 5% of OctanoylCarnosine

An oil-in-water cream containing 5% Octanoyl Carnosine, free of parabensand triethanolamine, was prepared as described above and contained thefollowing other components in order of descending predominance: water,octyldodecanol, decyl oleate, glyceryl stearate, propanediol, glycerin,stearic acid, Triticum vulgare (wheat) germ oil, ceteareth-20, cetylalcohol, Borago officinalis (borage) seed oil, dimethicone, Triticumvulgare (wheat) seed extract, myreth-3 myristate, ceteareth-12,tocopheryl acetate, tocopherol, cetearyl alcohol, cetyl palmitate,carbomer, disodium EDTA, sodium hydroxide, phenoxyethanol,ethylhexylglycerin, hexylene glycol, and caprylyl glycol.

Composition C: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, hydrogenatedpeanut oil, glycerin, cetearyl ethylhexanoate, cetearyl alcohol, PEG-8C12-18 alkyl ester, PPG-25-laureth-25, PEG-5 pentaerythrityl ether,hydroxyethylcellulose, cetyl alcohol, cetyl palmitate, glycerylstearate, sodium chloride, ascorbyl palmitate, glucose, simethicone,tocopheryl acetate, citric acid, ricinoleth-40, potassium chloride andmagnesium chloride. The cream further contained a mixture ofmetylparaben, propylparaben and imidazolidinyl urea for antimicrobialpreservation.

Composition D1: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, octyldodecanol,glyceryl stearate decyl oleate, glycerin, propylene glycol, Triticumvulgare (wheat germ oil), stearic acid, cetyl alcohol, ceteareth 20,myreth-3 myristate, ceteareth 12, cetearyl alcohol, cetyl palmitate,tocopheryl acetate, dimethicone, Borago officinalis (borage seed oil),carbomer, triethanolamine, methylparaben, propylparaben,glycosphingolipids (e.g., from Triticum vulgare (wheat) seed extract),disodium EDTA and BHT.

The cream further contained a mixture of phenoxyethanol, ethylparaben,butylparaben, methyl-isothiazolinone and methylchloroisothiazolinone forantimicrobial preservation.

Composition D2: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, octyldodecanol,glyceryl stearate decyl oleate, glycerin, propylene glycol, Triticumvulgare (wheat germ oil), stearic acid, cetyl alcohol, ceteareth 20,myreth-3 myristate, ceteareth 12, cetearyl alcohol, cetyl palmitate,tocopheryl acetate, dimethicone, Borago officinalis (borage seed oil),carbomer, triethanolamine, methylparaben, propylparaben,glycosphingolipids (e.g., from Triticum vulgare (wheat) seed extract),disodium EDTA and BHT.

Composition E: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, octyldodecanol,glyceryl stearate decyl oleate, glycerin, propylene glycol, Triticumvulgare (wheat germ oil), stearic acid, cetyl alcohol, ceteareth 20,myreth-3 myristate, ceteareth 12, cetearyl alcohol, cetyl palmitate,tocopheryl acetate, dimethicone, Borago officinalis (borage seed oil),carbomer, methylparaben, propylparaben, glycosphingolipids (e.g., fromTriticum vulgare (wheat) seed extract), and disodium EDTA. The creamfurther contained tocopherol, sodium hydroxide, and a mixture ofphenoxyethanol, ethylparaben, butylparaben for antimicrobialpreservation.

Composition F: Example of Oil-in-Water Cream Containing 0.013% TotalPeptides

An oil-in-water cream containing 0.013% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, caprylic/caprictriglyceride, C12-20 acid PEG-8 ester, butylene glycol, glycerin,saccharide isomerate, PEG-8, cetyl alcohol, caprylyl glycol, potassiumcetyl phosphate, carbomer, bisabolol, ascorbyl tetraisopalmitate,caffeine, disodium EDTA, phospholipids, glycyrrhetinic acid, sodiumhyaluronate, sodium polyacrylate, citric acid, propylparaben,tocopherol, beech tree bud extract (fagus sylvatica extract), palm oil(Elaeis guineensis), tocotrienols, ascorbyl palmitate, squalene,ascorbic acid and phytosterols. The cream further contained a mixture ofphenoxyethanol, methylparaben, butylparaben, and ethylparaben forantimicrobial preservation.

Composition G: Example of Oil-in-Water Cream Containing 0.013% TotalPeptides

An oil-in-water cream containing 0.013% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: WATER, C12-20 ACID PEG-8ESTER, PETROLATUM, CAPRYLIC/CAPRIC TRIGLYCERIDE, HYDROGENATEDPOLYISOBUTENE, GLYCERIN, SACCHARIDE ISOMERATE, HYDROXYETHYLACRYLATE/SODIUM ACRYLOYLDIMETHYL TAURATE COPOLYMER, BUTYLENE GLYCOL,ETHYLHEXYLGLYCERIN, ISOHEXADECANE, POTASSIUM CETYL PHOSPHATE, DIOSCOREAVILLOSA (WILD YAM) ROOT EXTRACT, BISABOLOL, ASCORBYL TETRAISOPALMITATE,CAFFEINE, DISODIUM EDTA, PHOSPHOLIPIDS, GLYCYRRHETINIC ACID, SODIUMHYALURONATE, ACRYLATES/C10-30 ALKYL ACRYLATE CROSSPOLYMER, CITRIC ACID,SODIUM CITRATE, SODIUM HYDROXIDE, TOCOPHEROL, BEECH TREE BUD EXTRACT(FAGUS SYLVATICA EXTRACT), PALM OIL (ELAEIS GUINEENSIS), TOCOTRIENOLS,SQUALENE, PHYTOSTEROLS, PHENOXYETHANOL, CHLORPHENESIN, POLYSORBATE 60,BENZYL ALCOHOL, BENZOIC ACID.

Composition H: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, caprylic/caprictriglyceride, C12-20 acid PEG-8 ester, coco-caprylate/caprate, butyleneglycol, dimethicone, phenyl trimethicone, biosaccharide gum-1, glycerin,cetyl alcohol, phenoxyethanol, saccharide isomerate, carbomer, potassiumcetyl phosphate, Borago officinalis (borage seed oil), ascorbyltetraisopalmitate, caprylyl glycol, methylparaben, disodium EDTA,chondrus crispus (carrageenan), sodium hyaluronate, Elaeis guineensis(palm) oil, tocotrienols, phytosterols, butylparaben, ethylparaben,PEG-8, isobutylparaben, propylparaben, tocopherol, citric acid, ascorbylpalmitate, squalene and ascorbic acid.

Composition I: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: water, ethylhexylmethoxycinnamate, C12-20 acid PEG-8 ester, caprylic/capric triglyceride,coco-caprylate/caprate, butylene glycol, butyl methoxydibenzoylmethane,cetyl alcohol, biosaccharide gum-1, glycerin, C12-15 alkyl benzoate,saccharide isomerate, phenoxyethanol, caprylyl glycol, titanium dioxide,potassium cetyl phosphate, carbomer, Borago officinalis seed oil,ascorbyl tetraisopalmitate, methylparaben, sodium hydroxide, disodiumEDTA, chondrus crispus (carrageenan), sodium hyaluronate, Elaeisguineensis (palm) oil, tocotrienols, phytosterols, butylparaben,aluminum stearate, polyhydroxystearic acid, ethylparaben, alumina,PEG-8, isobutylparaben, propylparaben, tocopherol, citric acid, BHT,ascorbyl palmitate, squalene and ascorbic acid.

Composition J: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5which provides additionally sun protection (e.g., of about SPF30)properties due to the presence of sunscreen actives including octinoxateand zinc oxide was prepared as described above and contained thefollowing other components: sunscreens actives: octinoxate (about 7.5%)and zinc oxide (about 7.3%). Remaining ingredients in order ofdescending predominance: Water, Caprylic/Capric Triglyceride,Hydrogenated C6-14 Olefin Polymers, Hexyldecanol, Glycerin, GlycerylStearate, PEG-100 Stearate, Potassium Cetyl Phosphate, Hydrogenated PalmGlycerides, Microcrystalline Cellulose, Camelia Sinensis Extract,Silica, Sodium Ascorbyl Phosphate, Tocopheryl Acetate, Squalane,Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer,Polyhydroxystearic Acid, Steareth-21, Melanin, Cetearyl Alcohol, SodiumHyaluronate, Polysorbate 60, Triethoxycaprylsilane, Disodium EDTA,Xanthan Gum, Styrene/Acrylates Copolymer, Phenoxyethanol, Methylparaben,Ethylparaben, Propylparaben.

Composition K: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5,free of parabens, triethanolamine and BHT, was prepared as describedabove and contained the following other components in order ofdescending predominance: water, octyldodecanol, decyl oleate, glycerylstearate, propanediol, glycerin, stearic acid, Triticum vulgare (wheat)germ oil, ceteareth-20, cetyl alcohol, Borago officinalis (borage) seedoil, dimethicone, Triticum vulgare (wheat) seed extract, myreth-3myristate, ceteareth-12, tocopheryl acetate, tocopherol, cetearylalcohol, cetyl palmitate, carbomer, disodium EDTA, sodium hydroxide,phenoxyethanol, ethylhexylglycerin, hexylene glycol, and caprylylglycol.

Composition L: Example of Oil-in-Water Serum Containing 0.02% TotalPeptides (Excluding TRIFLUORACETYL TRIPEPTIDE-2 which is Present in thisComposition as an Additional Peptide)

An oil-in-water serum containing 0.02% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: WATER, GLYCERIN,CAPRYLIC/CAPRIC TRIGLYCERIDE, HYDROXYETHYL ACRYLATE/SODIUMACRYLOYLDIMETHYL TAURATE COPOLYMER, PULLULAN, SQUALANE, AMINOPROPYLASCORBYL PHOSPHATE, BUTYLENE GLYCOL, SODIUM HYALURONATE, ACETYLGLUCOSAMINE, ARGININE, PROLINE, GLYCINE, GLUTAMINE, TRIFLUORACETYLTRIPEPTIDE-2, ALGAE EXTRACT, POLYSORBATE 60, DEXTRAN,ETHYLHEXYLGLYCERIN, ETHYLENE/ACRYLIC ACID COPOLYMER, DISODIUM EDTA,SODIUM HYDROXIDE, CHLORPHENESIN, PHENOXYETHANOL

Composition M: Example of Oil-in-Water Serum Containing 0.1% TotalPeptides (Excluding TRIFLUORACETYL TRIPEPTIDE-2 which is Present in thisComposition as an Additional Peptide)

An oil-in-water serum containing 0.1% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: WATER, GLYCERIN,CAPRYLIC/CAPRIC TRIGLYCERIDE, HYDROXYETHYL ACRYLATE/SODIUMACRYLOYLDIMETHYL TAURATE COPOLYMER, PULLULAN, SQUALANE, AMINOPROPYLASCORBYL PHOSPHATE, BUTYLENE GLYCOL, SODIUM HYALURONATE, ACETYLGLUCOSAMINE, ARGININE, PROLINE, GLYCINE, GLUTAMINE, TRIFLUORACETYLTRIPEPTIDE-2, ALGAE EXTRACT, POLYSORBATE 60, DEXTRAN,ETHYLHEXYLGLYCERIN, ETHYLENE/ACRYLIC ACID COPOLYMER, DISODIUM EDTA,SODIUM HYDROXIDE, CHLORPHENESIN, PHENOXYETHANOL.

Composition N: Example of Oil-in-Water Lotion Containing 0.01% TotalPeptides

An oil-in-water lotion containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5which provides additional anti-properties due to the presence of retinol(e.g., embedded in microcapsules for controlled release) was prepared asdescribed above and contained the following other components in order ofdescending predominance: Water, Caprylic/Capric Triglyceride, Glycerin,Cetearyl Alcohol, C10-30 Cholesterol/Lansterol Esters, CetylRicinoleate, Cetyl Alcohol, Dimethicone, Polysorbate 60, MethylMethacrylate/Glycol Dimethacrylate Crosspolymer, Benzyl Alcohol,Retinol, Ascorbic Acid, Ascorbyl Palmitate, Bisabolol, TocopherylAcetate, Cyclopentasiloxane, Cyclohexasiloxane, PEG-10 Soy Sterol,Stearic Acid, BHT, Propyl Gallate, Disodium EDTA, Magnesium AluminumSilicate, Phenoxyethanol, Polysorbate 20, Triethanolamine, andMethylparaben.

Composition O1: Example of a Serum Containing 0.01% Total Peptides

An serum containing 0.01% of the combination of Octanoyl Carnosine,Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5 andcontaining high levels of hyaluronic acid was prepared as describedabove and contained the following other components in order ofdescending predominance: WATER, SODIUM HYALURONATE, CITRIC ACID,PHENOXYETHANOL, SODIUM METHYLPARABEN, SODIUM BUTYLPARABEN, SODIUMPROPYLPARABEN, and SODIUM CHLORIDE.

Composition O2: Example of a Serum Containing 0.1% Total Peptides

An serum containing 0.1% of the combination of Octanoyl Carnosine,Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5 andcontaining high levels of hyaluronic acid was prepared as describedabove and contained the following other components in order ofdescending predominance: WATER, SODIUM HYALURONATE, CITRIC ACID,PHENOXYETHANOL, SODIUM METHYLPARABEN, SODIUM BUTYLPARABEN, SODIUMPROPYLPARABEN, and SODIUM CHLORIDE.

Composition P: Example of a Gel Containing 0.01% Total Peptides

A gel (e.g., hydrogel) containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5was prepared as described above and contained the following othercomponents in order of descending predominance: WATER (AQUA), BUTYLENEGLYCOL, GLYCERIN, CARBOMER, PEG-8, CAPRYLYL GLYCOL, CARRAGEENAN(CHONDRUS CRISPUS), PHENOXYETHANOL, DISODIUM EDTA, METHYLPARABEN,BUTYLPARABEN, SODIUM HYALURONATE, ETHYLPARABEN, SODIUM POLYACRYLATE,ISOBUTYLPARABEN, and PROPYLPARABEN.

Composition Q: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5which provides additionally skin lightening properties due to thepresence of skin lighting agents (as disclosed in WO 2010/083368) wasprepared as described above and contained the following othercomponents:

INGREDIENT INCI % BY NO. PHASE (TRADE NAME) DESIGNATION SUPPLIER WEIGHT1 A DEIONIZED WATER WATER (AQUA) 63.30 2 A NA₂EDTA DISODIUM EDTAAKZO/DEWOLF 0.100 3 A KELTROL CG-T XANTHAN GUM CP KELCO/ 0.300 UNIVAR 4B LIPOWAX D CETEARYL LIPO 8.250 ALCOHOL CETEARETH-20 5 B LIPO GMS 450GLYCERYL LIPO 6.000 STEARATE 6 B CERAPHYL 230 DIISOPROPYL ISP SUTTON5.000 ADIPATE 7 B DC TORAY FZ-3196 CAPRYLYL DOW 3.000 METHICONE CORNING/UNIVAR 8 B DC 200 FLUID 100 CST DIMETHICONE DOW 1.000 CORNING/ UNIVAR 9B LIPOVOL J SIMMONDSIA LIPO 1.000 CHINENSIS (JOJOBA) SEED OIL 10 B SHEABUTTER HMP BUTYROSPERMUM EARTH 1.000 PARKII (SHEA SUPPLIED BUTTER)PRODUCTS 11 B VITAMIN E ACETATE DL-ALPHA BASF/ 0.200 OIL (USP, FCC)TOCOPHERYL CHEMCENTRAL ACETATE 12 C DEIONIZED WATER WATER (AQUA) 0.10013 C ELESTAB CPN ULTRA CHLORPHENESIN COGNIS 0.300 PURE 14 C PHENOXETOLPHENOXYETHANOL CLARIANT 0.600 15 C SEPIWHITE MSH UNDECYLENOYL SEPPIC0.500 PHENYLALANINE 16 C SODIUM SODIUM DR. PAUL 3.000 GLYCEROPHOSPHATEGLYCEROPHOSPHATE LOHMANN (Ph. Eur. 6 Ed, Item# 500012045500) 17 CL-LEUCINE LEUCINE AJINOMOTO 1.000 18 C1 CITRIC ACID 50% CITRIC ACID PCI1.920 SOLUTION (TO pH 4.5-5.0) 19 C2 GLYCERIN 99.7% GLYCERIN ACME- 2.000(USP) HARDESTY 20 C2 SYMWHITE 377 PHENYLETHYL KAH/SYMRISE 0.500RESORCINOL 21 C2 VITAGEN AMINOPROPYL BASF 0.500 ASCORBYL PHOSPHATE 22 DSIMULGEL INS 100 HYDROXYETHYL SEPPIC 0.420 ACRYLATE/SODIUMACRYLOYLDIMETHYL TAURATE COPOLYMER ISOHEXADECANE POLYSORBATE 60 TOTAL99.99

Such compositions were generally prepared in a clean and sanitizedstainless steel vessel as described herein below:

-   PHASE A: DISPERSE KELTROL IN WATER, MIX UNTIL ALL HYDRATES; ADD    REMAINING PHASE A INGREDIENTS, HEAT TO ABOUT 75° C. WHILE MIX UNTIL    ALL DISSOLVES.-   PHASE B: COMBINE PHASE B INGREDIENTS IN A SEPARATE VESSEL AND MIX    WHILE HEATING TO 75° C.; ONCE ALL WAXES MELT AND PHASE IS AT TEMP    AND UNIFORM, SLOWLY ADD TO PHASE A; COOL TO 35° C.-   PHASE C: COMBINE PHASE C INGREDIENTS WITH MECHANICAL STIRRING UNIT    AND MIX WITH MODERATE AGITATION-   PHASE C1: USE PHASE C1 TO ADJUST pH OF PHASE C TO 4.0-4.5-   PHASE C2: COMBINE PHASE C2 AND MIX WHILE HEATING SLIGHTLY TO 40° C.;    CONTINUE MIXING UNTIL POWDERS DISSOLVE THEN ADD TO PHASE C; ADD    PHASE C TO BATCH WITH MODERATE AGITATION-   PHASE D: ADD PHASE D TO BATCH, MIX UNTIL UNIFORM; HOMOGENIZE THE    BATCH AT 3500 RPM FOR 5 MINUTES; SWITCH TO IMPELLER MIXING; COOL TO    ROOM TEMPERATURE.

Composition R: Example of Oil-in-Water Cream Containing 0.01% TotalPeptides

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5which provides additionally skin lightening properties due to thepresence of skin lighting agents including hydroquinone (as disclosed inWO 2010/083368) was prepared as described above and contained thefollowing other components:

INGREDIENT INCI % BY NO. PHASE (TRADE NAME) DESIGNATION SUPPLIER WEIGHT1 A DEIONIZED WATER WATER (AQUA) 59.670 2 A NA₂EDTA DISODIUM EDTA AKZO0.100 3 A KELTROL CG-T XANTHAN GUM CP KELCO 0.300 4 A ELESTAB CPN ULTRACHLORPHENESIN COGNIS 0.300 PURE 5 A PHENOXETOL PHENOXYETHANOL CLARIANT0.600 6 A SEPIWHITE MSH UNDECYLENOYL SEPPIC 0.500 PHENYLALANINE 7 ASODIUM SODIUM DR. PAUL 3.000 GLYCEROPHOSPHATE GLYCEROPHOSPHATE LOHMANN(Ph. Eur. 6 Ed, Item# 500012045500) 8 A L-LEUCINE LEUCINE AJINOMOTO1.000 9 B LIPOWAX D CETEARYL ALCOHOL LIPO 6.000 CETEARETH-20 10 B LIPOGMS 450 GLYCERYL STEARATE LIPO 6.000 11 B CERAPHYL 230 DIISOPROPYL ISPSUTTON 3.000 ADIPATE 12 B DC TORAY FZ-3196 CAPRYLYL DOW 3.000 METHICONECORNING 13 B DC 200 FLUID 100 CST DIMETHICONE DOW 1.000 CORNING/ UNIVAR14 B LIPOVOL J SIMMONDSIA LIPO 1.000 CHINENSIS (JOJOBA) SEED OIL 15 BSHEA BUTTER HMP BUTYROSPERMUM EARTH 1.000 PARKII (SHEA SUPPLIED BUTTER)PRODUCTS 16 B VITAMIN E ACETATE DL-ALPHA BASF/ 0.200 OIL (USP, FCC)TOCOPHERYL CHEMCENTRAL ACETATE 17 C CITRIC ACID 50% CITRIC ACID PCI1.920 SOLUTION (TO pH 4.5-5.0) 18 D EASTMAN ™ HYDROQUINONE EASTMAN/4.000 HYDROQUINONE CHEMPOINT (USP GRADE) 19 E SODIUM SODIUM UPI 0.400METABISULFITE METABISULFITE (NF/FCC) 20 F GLYCERIN 99.7% GLYCERIN ACME-2.000 (USP) HARDESTY 21 F SYMWHITE 377 PHENYLETHYL KAH/SYMRISE 0.500RESORCINOL 22 F VITAGEN AMINOPROPYL BASF 0.500 ASCORBYL PHOSPHATE 23 GSIMULGEL INS 100 HYDROXYETHYL SEPPIC 4.000 ACRYLATE/SODIUMACRYLOYLDIMETHYL TAURATE COPOLYMER ISOHEXADECANE POLYSORBATE 60 TOTAL99.99

Such compositions were generally prepared in a clean and sanitizedstainless steel vessel, which was suitable for blending productscontaining hydroquinone, as described herein below:

-   PHASE A: DISPERSE KELTROL IN WATER, MIX UNTIL ALL HYDRATES;    -   ADD EDTA, MIX UNTIL ALL DISSOLVES;    -   ADD REMAINING PHASE A INGREDIENTS, HEAT TO 75° C. WHILE MIX        UNTIL ALL DISSOLVES.-   PHASE B: COMBINE PHASE B INGREDIENTS, HEAT TO 75° C., MIX UNTIL ALL    MELTED AND UNIFORM;    -   WHEN BOTH PHASE A AND PHASE B AT 75° C., ADD PHASE B INTO PHASE        A WITH AGITATION MIX FOR 10 MINUTES, START COOLING TO 50° C.-   PHASE C: ADJUST pH WITH PHASE C TO pH 4.5-5.0, COOL TO 45° C.-   PHASE D: ADD PHASE D TO BATCH WITH MIX, MIX UNTIL ALL DISSOLVES AND    UNIFORM.-   PHASE E: ADD PHASE E TO BATCH WITH MIXING, MIX UNTIL ALL DISSOLVES.-   PHASE F: COMBINE PHASE F INGREDIENTS, SLIGHTLY HEAT AND MIX UNTIL    ALL DISSOLVES, ADD TO THE BATCH.-   PHASE G: ADD PHASE G TO BATCH, MIX UNTIL UNIFORM; HOMOGENIZE THE    BATCH AT 3500 RPM FOR 5 MINUTES, SWITCH TO IMPELLER MIXER, MIX;    -   ADJUST pH WITH PHASE C TO pH 4.5-5.0 IF NECESSARY.

Composition S: Example of oil-in-water serum containing 0.01% totalpeptides

An oil-in-water serum containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5which provides additionally anti-rosacea or skin redness reducingproperties due to the presence of antimicrobial peptide sequesteringagents such as the sodium salt of dextran sulfate (as disclosed in WO2011/109469) was prepared as described above and contained the followingother components:

% by Ingredient weight Phase (Trade Name) INCI Name Supplier(s) (% w) AWater Water (Aqua) 60.03 A Na2EDTA Disodium EDTA Akzo/DeWolf 0.1 AKeltrol CG-SFT Xanthan Gum CP Kelco/ 0.25 Univar A Structure XLHydroxypropyl National Starch 1.0 Starch Phosphate A Simulgel INS 100Hydroxyethyl Seppic 1.0 Acrylate/Sodium Acryloyldimethyl TaurateCopolymer (and) Isohexadecane (and) Polysorbate-60 B Water 15 BOristract CF Caffeine Orient Stars 1.5 B Dextran Sulfate Dextran SodiumMP Biomedical/ 0.5 Sodium Salt (av. Sulfate Spectrum M.W. about 8000) BAjidew ZN-100 Zinc PCA Ajinomoto 1 B1 Elestab CPN Ultra ChlorphenesinCognis 0.2 Pure B1 Glycerin 99.7% Glycerin Acme-Hardesty 15 B1Phenoxetol Phenoxyethanol Clariant/ 0.5 Essential Ingredients CStructure XL Hydroxypropyl National Starch 1.5 Starch Phosphate CSimulgel INS 100 Hydroxyethyl Seppic 0.41 Acrylate/SodiumAcryloyldimethyl Taurate Copolymer (and) Isohexadecane (and)Polysorbate-60 D DC Toray FZ-3196 Caprylyl Dow Corning/ 2 MethiconeUnivar

Such compositions were generally prepared in a clean and sanitizedstainless steel vessel as described herein below:

Phase A: Dissolve Na2EDTA into agitating Phase A water. Mix untiluniform. Sprinkle Keltrol slowly into batch. Mix until fully hydrated.Sprinkle Structure XL into agitating Phase A water. Mix until fullydispersed, and homogenize at 3500 RPM for 5-6 minutes. Add Simulgel INS100 and mix until uniform, then homogenize for about 4 minutes at 3500RPM.Phase B: Combine Phase B ingredients, one by one in a separate vesselwhile heating to 50-53° Celsius (not higher than 55° Celsius).Phase B1: In a separate vessel combine Phase B1 ingredients and heat to40° Celsius. Mix until powder is dispersed. Add Phase B1 to Phase B andmix until clear. Cool to 30° Celsius and add combined Phase B/B1 tobatch. Mix until uniform.Phase C: Add Phase C to batch one by one to raise viscosity, homogenizeafter adding the Structure X1 and again after adding the Simulgel INS100.Phase D: Add Phase D ingredients to batch and mix until uniform.

Example 5: Results of In Vitro Tests on Examples According to theInvention

In Vitro Test 1: Stimulation of Gene Expression of Extracellular MatrixComponents Including but not Limited to Collagens I and III by OctanoylCarnosine

Normal human dermal fibroblasts (NHDF); used at the 8^(th) passage, werecultured in culture medium (DMEM supplemented with 10% fetal calf serum,2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin) in24-well plates for 48 h at 37° C. and 5% CO₂ with a renewal of culturemedium after 24 h. The culture medium was then removed, replaced byassay medium (DMEM culture medium supplemented with 1% fetal calf serum,2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin) and thecells were further incubated for 24 h. The cells were then treated withthe test peptides, or not (control), or with the reference TGF-beta 1and incubated for 24 h. All experimental conditions were performed4-times (n=4). At the end of the incubation, the cells were washed inPBS solution and immediately frozen at about −80° C. The expression ofthe markers was analyzed using quantitative real time polymerase chainreaction (RT-qPCR) method on mRNA extracted from the cell monolayers foreach treatment; whereby the replicates were pooled before RNAextraction. Analysis of gene expression was performed in duplicate (n=2)using a PCR array. Total RNA was extracted from each sample usingTriPure Isolation Reagent® according to the supplier's instructions. Theamount and quality of RNA were evaluated using a lab-on-a-chipBioanalyzer (Agilent Technologies). Potential contaminant traces ofgenomic DNA were removed using the DNAfree system (Ambion). Thereverse-transcription of mRNA was conducted in presence of oligo(dT) andSuperscript II reverse-transcriptase. Quantification of cDNA wasperformed using Nanovue (GE Healthcare) and cDNA was adjusted.

The PCRs were performed using the LightCycler® system (Roche MolecularSystem Inc.) according to the supplier's instructions. This systemallowed rapid and powerful PCRs, after determining analysis conditionsof the test primers. The reaction mix (10 μl final) was added asfollows: 2.5 μl of cDNA, primers forward and reverse; and reagent mixcontaining taq DNA polymerase, SYBR Green I, and MgCl₂.

The incorporation of the fluorescence in amplified DNA was continuouslymeasured during the PCR cycles. This resulted in a “fluorescenceintensity” versus “PCR cycle” plot allowing the evaluation of a relativeexpression (RE) value for each marker. The value selected for REcalculations is the “output point” (Ct) of the fluorescence curve. For aconsidered marker, the highest is the cycle number; the lowest is themRNA quantity. The RE value was expressed in arbitrary units (AU)according to the formula: (½^(number of cycles))×10⁶.

This evaluation was realized with non-cytotoxic concentrations of thetest peptides. Cytotoxicity of the test peptides in the assay medium waspreviously determined in NHDF by the MTT reduction assay andmorphological observations with help of microscope after a 24 hincubation time.

This test surprisingly revealed that the peptide Octanoyl Carnosine isable to significantly stimulate extracellular matrix components otherthan only collagen I. It was discovered that Octanoyl Carnosine at 300ppm (0.03%) stimulates the formation of collagen I (alpha 1) by 38%,collagen III (alpha 1) by 120%, collagen V (alpha 1) by 35%, collagen VI(alpha 1) by 46%, collagen VII (alpha 1) by 97%, collagen XVI (alpha 1)by 91%, elastin by 51%, laminin (alpha 4) by 20%, hyaluronan synthase 2by 23%, fibrillin 1 by 23%, and heparan sulfate protoglycan 2 by 79%.

Even more unexpectedly, this test surprisingly revealed that the peptideOctanoyl Carnosine is able to stimulate collagen III significantly morethan collagen I; collagen III (alpha 1) formation was stimulated by120%, whereas collagen I (alpha 1) only by 38%. This property ofOctanoyl Carnosine was unexpected as other peptides which stimulateformation of both collagen I and collagen III generally stimulatecollagen I significantly more than collagen III. For example, as alsodemonstrated in this test, beta-alanyl-histidine (the natural carnosine)at 300 ppm (0.03%) stimulates formation of collagen III only by 24% butstimulates collagen I at 28%. Furthermore, TGF-beta 1 (10 ng/ml) wasshown to stimulate formation of collagen III by 164% but at the sametime stimulates collagen I by 245%.

For the interpretation of the data related to the stimulation of theformation of extracellular matrix components as determined during the invitro experiments, a “significant” stimulation of the formation ofextracellular matrix components means a stimulation of at least 20% morethan measured using a control material under identical experimentalconditions. The control material is the identical material (here: assaymedium) as used when testing the active (here: peptide(s)) but withoutthe active(s).

Consequently, compositions containing Octanoyl Carnosine (or one or morederivatives thereof) according to the present invention have thereforegreat potential in the field of cosmetics, dermatology, wound healing,and any other areas in need of treatments for conditions, disorders anddiseases where extracellular matrix components (including, but notlimited to, collagen III) are altered. In addition, such compositionscan be used for maintaining healthy skin, skin rejuvenation, scarlesswound healing, restoration of damaged skin and mucosa, as well as forthe treatment of atrophy of any human tissue including, but not limitedto, vulvovaginal atrophy.

In contrast, the prior art discloses other peptides significantlystimulating both collagen I and collagen III formation, but theystimulate collagen I more than collagen III.

In Vitro Test 2: Stimulation of Hyaluronic Acid—Combination of OctanoylCarnosine with Other Peptides

Normal human dermal fibroblasts (NHDF); used at the 8^(th) passage, werecultured in culture medium (DMEM supplemented with 10% fetal calf serum,2 mM L-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin) in96-well plates for 24 h at 37° C. and 5% CO₂.

Afterwards, the culture medium was removed and replaced by assay medium(DMEM culture medium supplemented with 2% fetal calf serum, 2 mML-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin) containingthe test peptides or not (control). The cells were then incubated for 72h. All experimental conditions were performed 3-times (n=3). At the endof the incubation, culture supernatants were collected and immediatelyfrozen at about −80° C.

Hyaluronic acid was quantified in the culture supernatant using theDuoset Hylaluronan ELISA kit (R&D Systems Ref. DY3614, 0.37 ng/ml as lowdetection limit) according to the provider's specifications.

This evaluation was realized with non-cytotoxic concentrations of thetest peptides. Cytotoxicity of the test peptides in the assay medium waspreviously determined in NHDF by the MTT reduction assay andmorphological observations with help of microscope after a 72 hincubation time.

This test surprisingly revealed that the combination of OctanoylCarnosine with both Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at a weightratio of 4 parts Octanoyl Carnosine, 1 part Palmitoyl-GHK, and 5 partsGEKG (SEQ ID NO: 1) stimulates synthesis of hyaluronic acidsignificantly more than at other ratios. We discovered that acombination of 100 ppm (0.01%) Octanoyl Carnosine, 25 ppm (0.0025%)Palmitoyl-GHK and 125 ppm (0.0125%) GEKG (SEQ ID NO:1) significantlystimulates the synthesis of hyaluronic acid by 64%.

This significant stimulation of the synthesis of hyaluronic acid usingOctanoyl Carnosine when combined with Palmitoyl-GHK and GEKG (SEQ IDNO: 1) at a weight ratio of 4:1:5 was unexpected, as other weight ratiosdid not produce similar stimulation of hyaluronic acid synthesis.

For instance, this test revealed that a combination at the same totalconcentration of peptides (250 ppm) at a weight ratio of 1:2:2 (OctanoylCarnosine:Palmitoyl-GHK:GEKG (SEQ ID NO:1)) only non-significantlystimulated the synthesis of hyaluronic acid by 8%. Likewise,combinations at a weight ratio of 1:1:2 (tested at total peptideconcentrations of 132 ppm and 400 ppm), 1:3:1 (166 and 500 ppm), and1:30:1 (320 ppm) non-significantly stimulated the synthesis ofhyaluronic acid by less than 20%.

Even more surprisingly, this test also revealed that the combination ofOctanoyl Carnosine with both Palmitoyl-GHK and GEKG (SEQ ID NO: 1) at aweight ratio of 4 parts Octanoyl Carnosine, 1 part Palmitoyl-GHK, and 5parts GEKG (SEQ ID NO: 1) stimulates synthesis of hyaluronic acid in asynergistic manner. Specifically, a combination of 100 ppm (0.01%)Octanoyl Carnosine, 25 ppm (0.0025%) Palmitoyl-GHK and 125 ppm (0.0125%)GEKG (SEQ ID NO: 1) significantly stimulates the synthesis of hyaluronicacid by 64%. However, Octanoyl Carnosine alone at same concentration(100 pm) did not stimulate the synthesis of hyaluronic acid (the resultsshow a −5% increase, which, in fact, means a non-significant inhibitionof hyaluronic acid synthesis); Palmitoyl-GHK alone (at 10 or 30 ppm)non-significantly stimulated hyaluronic acid synthesis by only up to11%; and GEKG (SEQ ID NO:1) alone (at 100 or 300 ppm) significantlystimulated hyaluronic acid synthesis but only by up to 25%.

Consequently, if the effect of the combination of these three peptides(or any derivatives thereof) was additive, one would only expect onlyabout a 30-40% increase in hyaluronic acid synthesis (correspondingapproximately to the sum of −5% plus 11% plus 25%). However,unexpectedly, the combination of Octanoyl Carnosine, Palmitoyl-GHK andGEKG (SEQ ID NO: 1) at the weight ratio of 4:1:5 resulted in astimulation hyaluronic acid formation by 64%, which demonstrates that acombination of Octanoyl Carnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1)at the weight ratio of 4:1:5 provides synergistic effects (i.e., totaleffect of the combination of the three peptides is greater than the sumof the effects of the three peptides alone) in the stimulation ofhyaluronic acid formation.

Therefore, compositions containing Octanoyl Carnosine combined withPalmitoyl-GHK and GEKG (SEQ ID NO:1) (or one or more derivativesthereof) at the weight ratio of 4:1:5 according to the present inventionhave therefore great potential in the field of cosmetics, dermatology,wound healing, and any other area in the need of treatments forconditions, disorders and diseases where hyaluronic acid is altered. Inaddition, such compositions can be used for maintaining healthy skin,skin rejuvenation, scarless wound healing, restoration of damaged skinand mucosa, as well as for the treatment of atrophy of any human tissueincluding but not limited to vulvovaginal atrophy.

Example 6: Results of Clinical Tests on Examples According to theInvention

The following clinical tests with compositions according to the presentinvention illustrate a series of examples of the different uses of thecompositions and demonstrate the suitability of the compositions fortopical application in the field of cosmetics, dermatology, woundhealing, and any other areas in need of treatments for conditions,disorders and diseases where extracellular matrix components (including,but not limited to, collagen III) are altered; and in particular formaintaining healthy skin, skin rejuvenation, scarless wound healing,restoration of damaged skin and mucosa, as well as for the treatment ofatrophy of any human tissue including but not limited to vulvovaginalatrophy.

Clinical Test 1: Anti-Aging Study

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5(Composition D2; prepared as described in Example 4) was studied inhumans for maintaining healthy skin and skin rejuvenation. The humansubjects applied the cream twice daily to their face over a period ofsix months. They were asked to apply the cream as they would normallyuse a cosmetic facial product (e.g., facial moisturizer). Evaluationsfor safety and effectiveness were performed before the treatment withthe cream started (baseline), and after 1, 2, 3 and 6 months,respectively. The evaluations included the assessment of the subject'sface by trained investigators for fine lines and coarse wrinkles in thepen-orbital area, fine lines and coarse wrinkles in the pen-oral area,tactile roughness, mottled hyperpigmentation, redness andtelangiectasia, skin pores and pore size, skin tone, and for global skindamage using visual severity scores. The evaluations for effectivenessfurther included clinical photography of the face (under standardizedconditions including position of the subject and lighting),quantification of the skin roughness and wrinkle depths of theperi-orbital skin area using the PRIMOS-3D System (GF Messtechnik GmbH,Germany; utilizing standardized measuring and lighting conditions in the‘Overlay’ mode), as well as the measurement of skin elasticity of theupper cheek area with a Cutometer® MPA580 device (Courage & Khazaka,Koln, Germany; probe aperture 2 mm) after the subjects have been in aseated position in the environmentally-controlled room for at least 30minutes. The assessment of erythema (redness), edema (swelling),dryness, and peeling (desquamation) was performed to determine thesafety of the cream. For this purpose, the investigator used thefollowing 5-point scale: 0=none, 1=mild, 2=moderate, 3=marked, 4=severe.Furthermore, adverse events were recorded during the entire studyduration. In a subset of subjects, a 3 mm punch biopsy was taken fromthe pre-auricular skin area under local anesthesia (i.e., up to 1 cclidocaine with epinephrine given intradermally) performed by the studydoctor before treatment and at the end of the six month treatmentperiod. The sequential biopsies were nearly adjacent to each other. Thebiopsy specimens were fixed in formaldehyde, embedded in standardembedding medium, and then evaluated for changes in epidermal and dermalstructure (and for changes of the extracellular matrix in particular) byhistology. In addition, the subjects reported about diverse attributesof the cream (benefits, improvements, tolerability, cosmetic feel,satisfaction, etc.) using questionnaires.

The following results were obtained in a study which included females ofskin types I to III with at least moderate signs of facial wrinkles.Peri-orbital wrinkles improved (by at least 1 unit) in 35% of thesubjects after 1 month (from 2.9±0.5 before treatment to 2.6±0.6;mean+SD, n=20 subjects), 71% after 3 months (2.1±0.6; n=17), and 88%after 6 months (1.8±0.4; n=17). Peri-oral wrinkles improved (by at least1 unit) in 30% of the subjects after 1 month (from 2.7±0.6 beforetreatment to 2.4±0.6; mean+SD, n=20), 47% after 3 months (2.2±0.5;n=17), and 71% after 6 months (1.7±0.5; n=17). The product was welltolerated.

This study clearly demonstrated that an oil-in-water cream containing0.01% of the combination of Octanoyl Carnosine, Palmitoyl-GHK and GEKG(SEQ ID NO: 1) at a weight ratio 4:1:5 rejuvenates aged skin. Thecomposition was further well tolerated in all subjects and did not causeany adverse events.

Clinical Test 2: Skin Restoration Studies after Cosmetic andDermatological Procedures

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5(Composition E; prepared as described in Example 4) was studied inhumans for enhancing the restoration of skin after cosmetic anddermatological procedures such as chemical skin peels, skin abrasiontreatments, skin laser treatments, skin light treatments, skinradiofrequency treatments, skin ultrasound, cold or hot treatments ofskin, non-surgical face lifts; or combinations thereof. The subjectswere asked to apply the cream once to six-times daily to the affectedskin area until at least the skin has healed. This study involvingdifferent clinical case trials demonstrated that an oil-in-water creamcontaining 0.01% of the combination of Octanoyl Carnosine, Palmitoyl-GHKand GEKG (SEQ ID NO: 1) at a weight ratio 4:1:5 enhances the restorationprocess of skin (e.g., decrease of time to heal, reduction of severityof side effects due to procedure, reduction of skin inflammation,decrease of time for restoring normal skin barrier, decrease of time forre-epithelialization, increase of hydration, decrease of time to appearnormal again, help to improve appearance, help to reduce signs of skinaging, contribution to increase collagen III formation, does not resultin scar formation, etc.) after cosmetic and dermatological procedures.The composition was further well tolerated.

A serum containing 0.5% of Octanoyl Carnosine (Composition A3; preparedas described in Example 4) was studied in humans for enhancing therestoration of skin after cosmetic and dermatological procedures. Thesubjects were asked to apply the serum once to six-times daily to theaffected skin area until at least the skin has healed. This studyinvolving different clinical case trials demonstrated that a serumcontaining 0.5% of Octanoyl Carnosine enhances the restoration processof skin after cosmetic and dermatological procedures. The compositionwas further well tolerated.

Clinical Test 3: Wound Healing Studies

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5(Composition D2; prepared as described in Example 4) was studied inhumans for wound healing of surgical wounds, accidental wounds, skinulcers, and skin burns. The subjects were asked to apply the cream onceto six-times daily to the wounded area until at least the wound hashealed. This study involving different clinical case trials demonstratedthat an oil-in-water cream containing 0.01% of the combination ofOctanoyl Carnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weightratio 4:1:5 accelerates wound closure and helps skin heal without scarformation. The composition was further well tolerated.

A serum containing 0.5% of Octanoyl Carnosine (Composition A3; preparedas described in Example 4) was studied in humans for wound healing ofsurgical wounds, accidental wounds, skin ulcers, and skin burns. Thesubjects were asked to apply the serum once to six-times daily to thewounded area until at least the wound has healed. This study involvingdifferent clinical case trials demonstrated that a serum containing 0.5%of Octanoyl Carnosine accelerates wound closure and helps skin healwithout scar formation. The composition was further well tolerated.

Clinical Test 4: Skin Atrophy Studies

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5(Composition E; prepared as described in Example 4) was studied inhumans with skin atrophy due to long-term topical corticosteroids use,or respectively, after menopause in women. The subjects were asked toapply the cream once to six-times daily for at least four weeks to theaffected skin area. This study involving different clinical case trialsdemonstrated that an oil-in-water cream containing 0.01% of thecombination of Octanoyl Carnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1)at a weight ratio 4:1:5 reduce the severity of skin atrophy and helpsrestore a normal skin barrier. The composition was further welltolerated.

A serum containing 0.5% of Octanoyl Carnosine (Composition A3; preparedas described in Example 4) was studied in humans with skin atrophy dueto long-term topical corticosteroids use, or respectively, aftermenopause in women. The subjects were asked to apply the serum once tosix-times daily for at least four weeks to the affected skin area. Thisstudy involving different clinical case trials demonstrated that a serumcontaining 0.5% of Octanoyl Carnosine reduces the severity of skinatrophy and helps restore a normal skin barrier. The composition wasfurther well tolerated.

Clinical Test 5: Vulvovaginal Atrophy Studies

An oil-in-water cream containing 0.01% of the combination of OctanoylCarnosine, Palmitoyl-GHK and GEKG (SEQ ID NO:1) at a weight ratio 4:1:5(Composition K; prepared as described in Example 4) was studied in womenwith vulvovaginal atrophy. In those women, vulvovaginal atrophy was dueto menopause (either naturally occurring, or surgically induced), use oforal contraceptives, when undergoing breast cancer treatments (e.g.,including taking aromatase inhibitors), or due to other causes alteringtheir human sex hormone levels. The women were asked to apply the creamonce to four-times daily for at least four weeks to their vulva by usingtheir index finger. This study involving different clinical case trialsdemonstrated that an oil-in-water cream containing 0.01% of thecombination of Octanoyl Carnosine, Palmitoyl-GHK and GEKG (SEQ ID NO: 1)at a weight ratio 4:1:5 reduces the severity of the symptoms associatedwith vulvovaginal atrophy (e.g., dryness of vulva, feel of rawness,discomfort, pain with sex, itch, etc.) as well as reduces the severityof vulvovaginal atrophy; in particular for the vulva.

Clinical Test 6: Studies in Other Conditions, Disorders and Diseaseswhere Extracellular Matrix Components are Altered

Compositions according to the present invention were also studied forthe treatment of other conditions, disorders and diseases whereextracellular matrix components are altered including atopic dermatitis,eczema, scars and keloids, atrophie blanche, vulvar lichen sclerosus,epidermolysis bullosa, Ehlers-Danlos syndromes, and the Marfan syndrome.The subjects were asked to apply the compositions between once tosix-times daily for at least four weeks to the affected skin area. Thisstudy involving different clinical case trials demonstrated that thecompositions help reduce the symptoms and the severity of thoseconditions, disorders and diseases. The composition was generally welltolerated.

EQUIVALENTS

The details of one or more embodiments of the invention are set forth inthe accompanying description above. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. All patents and publications citedin this specification are incorporated by reference.

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto.

I claim:
 1. A method of stimulating the formation of collagen III inskin or mucosa of a patient, comprising topically administering aneffective amount of a composition comprising N-Octanoyl carnosine to thepatient, wherein the composition stimulates the formation of collagenIII to a higher degree than the formation of collagen I.
 2. The methodof claim 1, wherein the composition is administered to the patient inthe form of a cosmetic, a personal care product, a feminine careproduct, a hygiene product, a dermatology product, a pharmaceuticalpreparation, a medicament, or any combination thereof.
 3. The method ofclaim 1, wherein the composition is administered topically to the skinor mucosa of the patient.
 4. The method of claim 3, wherein thecomposition stimulates the formation of collagen III by at least about120% and the formation of collagen I by at least about 38%.
 5. Themethod of claim 3, wherein the composition is administered to a) one ormore areas of skin selected from the group consisting of face, neck,neckline, decollete, scalp, hand, palm, arm, leg, foot, sole, chest,breast, back, abdomen, buttock, vulva, penis, scrotum, and anus; or b)one or more mucosal areas selected from the group consisting of eye,mouth, nose, nipples, vulva, vagina, vaginal introitus, penis, andrectum.
 6. The method of claim 1, wherein the composition furthercomprises N-Palmitoyl-GHK and a tetra-peptide GEKG (SEQ ID NO:1),wherein the weight ratio of N-Octanoyl carnosine: N-Palmitoyl-GHK: GEKG(SEQ ID NO:1) is 4:1:5.
 7. The method of claim 1, wherein the methodresults in one or more of the following: improving the appearance ofaged skin, improving the state of aged skin, reducing the signs ofaging, reducing scarring of damaged skin, or enhancing the restorationof skin after cosmetic and dermatological procedures.
 8. The method ofclaim 1, wherein the composition further stimulates the formation ofelastin, laminin, hyaluronan synthase 2, fibrillin 1, and/or heparansulfate proteoglycan.